Leishmania antigens for use in the therapy and diagnosis of leishmaniasis

ABSTRACT

Compositions and methods for preventing, treating and detecting leishmaniasis and stimulating immune responses in patients are disclosed. The compounds provided include polypeptides that contain at least an immunogenic portion of one or more Leishmania antigens, or a variant thereof. Vaccines and pharmaceutical compositions comprising such polypeptides, or polynucleotides encoding such polypeptides, are also provided and may be used, for example, for the prevention and therapy of leishmaniasis, as well as for the detection of Leishmania infection.

REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of U.S. patentapplication Ser. No. 09/639,206 filed Aug. 14, 2000, which is acontinuation-in-part of U.S. patent application Ser. No. 09/565,501filed May 5, 2000, which is a continuation-in-part of U.S. patentapplication Ser. No. 09/551,974 filed Apr. 14, 2000, which is acontinuation-in-part of U.S. patent application Ser. No. 09/183,861,filed Oct. 30, 1998, which is a continuation in part of U.S. patentapplication Ser. No. 09/022,765, filed Feb. 12, 1998, which is acontinuation-in-part of U.S. patent application Ser. No. 08/920,609,filed Aug. 27, 1997, which is a continuation-in-part of U.S. patentapplication Ser. No. 08/798,841, filed Feb. 12, 1997, which is acontinuation-in-part of U.S. patent application Ser. No. 08/533,669,filed Sep. 22, 1995, now U.S. Pat. No. 5,834,592.

TECHNICAL FIELD

[0002] The present invention relates generally to compositions andmethods for preventing, treating and detecting leishmaniasis, and forstimulating immune responses in patients. The invention is moreparticularly related to polypeptides comprising an immunogenic portionof a Leishmania antigen or a variant thereof, and to vaccines andpharmaceutical compositions comprising one or more such polypeptides.The vaccines and pharmaceutical compositions may be used, for example,for the prevention and therapy of leishmaniasis, as well as for thedetection of Leishmania infection.

BACKGROUND OF THE INVENTION

[0003] Leishmania organisms are intracellular protozoan parasites ofmacrophages that cause a wide range of clinical diseases in humans anddomestic animals, primarily dogs. In some infections, the parasite maylie dormant for many years. In other cases, the host may develop one ofa variety of forms of leishmaniasis. For example, the disease may beasymptomatic or may be manifested as subclinical visceral leishmaniasis,which is characterized by mild symptoms of malaise, diarrhea andintermittent hepatomegaly. Patients with subclinical or asymptomaticdisease usually have low antibody titers, making the disease difficultto detect with standard techniques. Alternatively, leishmaniasis may bemanifested as a cutaneous disease, which is a severe medical problem butis generally self- limiting, or as a highly destructive mucosal disease,which is not self-limiting. Finally, and most seriously, the disease maybe manifested as an acute visceral infection involving the spleen, liverand lymph nodes, which, untreated, is generally a fatal disease.Symptoms of acute visceral leishmaniasis include hepatosplenomegaly,fever, leukopenia, anemia and hypergammaglobulinemia.

[0004] Leishmaniasis is a serious problem in much of the world,including Brazil, China, East Africa, India and areas of the MiddleEast. The disease is also endemic in the Mediterranean region, includingsouthern France, Italy, Greece, Spain, Portugal and North Africa. Thenumber of cases of leishmaniasis has increased dramatically in the last20 years, and millions of cases of this disease now exist worldwide.About 2 million new cases are diagnosed each year, 25% of which arevisceral leishmaniasis. There are, however, no vaccines or effectivetreatments currently available.

[0005] Accurate diagnosis of leishmaniasis is frequently difficult toachieve. There are 20 species of Leishmania that infect humans,including L. donovani, L. chagasi, L. infantum, L. major, L.amazonensis, L. braziliensis, L. panamensis, L. mexicana, L. tropica,and L. guyanensis, and there are no distinctive signs or symptoms thatunambiguously indicate the presence of Leishmania infection. Parasitedetection methods have been used, but such methods are neither sensitivenor clinically practical. Current skin tests typically use whole orlysed parasites. Such tests are generally insensitive, irreproducibleand prone to cross-reaction with a variety of other diseases. Inaddition, the preparations employed in such tests are often unstable.Thus, there is a need for improved methods for the detection ofLeishmania infection.

[0006] Current experimental vaccines consisting of whole organisms havenot proven effective in humans. Accordingly, there remains a need in theart for vaccines to prevent leishmaniasis in humans and dogs, and forimproved therapeutic compositions for the treatment of leishmaniasis.

SUMMARY OF THE INVENTION

[0007] Briefly stated, the present invention provides compositions andmethods for preventing, treating and detecting leishmaniasis, as well asfor stimulating immune responses in patients. In one aspect,polypeptides are provided which comprise at least an immunogenic portionof a Leishmania antigen, or a variant of such an antigen that differsonly in conservative substitutions and/or modifications. In specificembodiments of the invention, the Leishmania antigen comprises an aminoacid sequence selected from the group consisting of SEQ ID NO: 2, 4, 20,22, 24, 26, 36-38, 41, 50-53, 82, 104, 106, 108, 110, 112 and 118-122,.DNA sequences encoding the above polypeptides, recombinant expressionvectors comprising these DNA sequences and host cells transformed ortransfected with such expression vectors are also provided.

[0008] In further aspects, the present invention provide fusion proteinscomprising Leishmania antigens, together with polynucleotides encodingsuch fusion proteins. In certain specific embodiments, such fusionproteins comprise an amino acid sequence selected from the groupconsisting of SEQ ID NO: 95, 98 and 99.

[0009] In related aspects, the present invention provides pharmaceuticalcompositions which comprise one or more of the polypeptides and/orfusion proteins described herein, or a polynucleotide encoding suchpolypeptides and fusion proteins, and a physiologically acceptablecarrier. Vaccines which comprise one or more such polypeptides, fusionproteins or polynucleotides, together with an immunostimulant are alsoprovided. In specific embodiments of these aspects, the Leishmaniaantigen has an amino acid sequence selected from the group consisting ofSEQ ID NO: 2, 4, 20, 22, 24, 26, 36-38, 41, 50-53, 82, 104, 106, 108,110, 112 and 118-122.

[0010] In still further related embodiments, the pharmaceuticalcompositions and vaccines comprise at least two different polypeptides,each polypeptide comprising an immunogenic portion of a Leishmaniaantigen having an amino acid sequence selected from the group consistingof sequences recited in SEQ ID NO: 2, 4, 6, 8, 10, 20, 22, 24, 26,36-38, 41, 50-53, 82, 104, 106, 108, 110, 112 and 118-122, and variantsthereof that differ only in conservative substitutions and/ormodifications. In other embodiments, the inventive pharmaceuticalcompositions comprise one or more of the inventive polypeptides incombination with a known Leishmania antigen.

[0011] In yet other related embodiments, the pharmaceutical compositionsand vaccines comprise soluble Leishmania antigens.

[0012] In another aspect, the present invention provides methods forinducing protective immunity against leishmaniasis in a patient,comprising administering to a patient a pharmaceutical composition orvaccine as described above.

[0013] In further aspects, methods and diagnostic kits are provided fordetecting Leishmania infection in a patient. The methods comprise: (a)contacting dermal cells of a patient with a pharmaceutical compositionas described above; and (b) detecting an immune response on thepatient's skin, therefrom detecting Leishmania infection in the patient.The diagnostic kits comprise: (a) a pharmaceutical composition asdescribed above; and (b) an apparatus sufficient to contact thepharmaceutical composition with the dermal cells of a patient.

[0014] In further aspects, the present invention provides methods forstimulating a cellular and/or humoral immune response in a patient,comprising administering to a patient a pharmaceutical composition orvaccine as described above.

[0015] In a related aspect, methods are provided for treating a patientafflicted with a disease responsive to IL-12 stimulation, comprisingadministering to a patient a pharmaceutical composition or vaccine asdescribed above.

[0016] These and other aspects of the present invention will becomeapparent upon reference to the following detailed description andattached drawings. All references disclosed herein are herebyincorporated by reference in their entirety as if each was incorporatedindividually.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017]FIG. 1 shows the stimulation of proliferation of T-cells obtainedfrom L. donovani-immunized BALB/c mice (represented by stimulationindex) by L. donovani-infected macrophages after incubation for 24, 48and 72 hours.

[0018]FIG. 2 illustrates representative HPLC profiles of peptidesisolated from MHC class II molecules of P388D1 macrophages. Panel Ashows peptides isolated from uninfected macrophages and panel B showspeptides isolated from L. donovani infected macrophages. The arrows inpanel B indicate peptide peaks present only in the infected macrophagepreparation.

[0019]FIG. 3 illustrates the expression and purification of theLeishmania antigen Ldp23 as a recombinant fusion protein. Panel A showsa Coomassie blue-stained SDS-PAGE gel of lysed E. coli without (lane 1)and with (lane 2) IPTG induction of Ldp23 expression. Arrow indicatesthe recombinant fusion protein. Panel B shows the fusion proteinfollowing excision from a preparative SDS-PAGE gel, electroelution,dialysis against PBS and analytical SDS-PAGE.

[0020]FIG. 4 presents a Northern blot analysis of total RNA preparedfrom L. donovani, L. major, L. amazonensis and L. pifanoi with a ³²Plabeled Ldp23 gene. 1, 2 and 3 refer to RNA obtained from promastigotesat the logarithmic growth phase, promastigotes at the stationary growthphase and amastigote forms, respectively.

[0021]FIG. 5 shows a Western blot analysis of L. donovani promastigoteantigens incubated with pre-immune rabbit serum (lane A) or withanti-Ldp23 rabbit antiserum (lane B).

[0022]FIG. 6 illustrates the surface expression of Ldp23 on live L.donovani promastigotes. The dotted line shows the indirectimmunofluorescence performed using pre-immune mouse serum and the solidline shows the result obtained with mouse anti-GST-Ldp23 antiserum.Fluorescence intensity was analyzed by FACScan.

[0023]FIG. 7 shows the stimulation of Leishmania-specific T-cellproliferation by Ldp23. The results are presented as relative cellnumber as a function of fluorescence intensity. T-cells (10⁵/well) werepurified from lymph nodes of BALB/c mice immunized in the foot pad withL. donovani promastigotes in CFA and were cultured with variousconcentrations of the purified recombinant Ldp23 in the presence of2×10⁵ Mitomycin C-treated normal BALB/c spleen mononuclear cells.Proliferation of T-cells was measured at 27 hours of culture. Values areexpressed as cpm and represent the mean of [³H]TdR incorporation oftriplicate cultures.

[0024]FIG. 8 illustrates Ldp23-induced cytokine production by lymph nodecells of BALB/c mice. Cultures were incubated with varying amounts ofLdp23 or Leishmania lysate, presented as μg/mL, and were assayed byELISA for the production of interferon-y (panel A) or interleukin-4(panel B), both of which are shown as ng/mL.

[0025]FIG. 9 shows the PCR amplification of cytokine mRNAs isolated frommucosal leishmaniasis (Panel A) and cutaneous leishmaniasis (panel B)patient PBMC before and after stimulation with representativepolypeptides of the present invention. Lanes O and - indicate the levelof PCR products at the initiation of culture and after 72 hours ofculture, respectively, in the absence of added polypeptide; lanes Lb,83a and 83b indicate the level of PCR products following culturing ofPBMC with L. braziliensis lysate, and the Leishmania antigens Lbhsp83aand Lbhsp83b, respectively.

[0026]FIG. 10 presents a comparison of the levels of interferon-γ (panelA) and TNF-α (panel B) in the supernatants of 72 hour PBMC cultures fromLeishmania-infected and control individuals in response to stimulationwith parasite lysate or the indicated polypeptides.

[0027]FIG. 11 illustrates the levels of IL-10 p40 (in pg/mL) in thesupernatant of PBMC cultures from L. braziliensis-infected individualsand uninfected controls 72 hours following stimulation with parasitepromastigote lysate (Lb), Lbhsp83a or Lbhsp83b.

[0028]FIG. 12 presents the reactivities of sera from L. braziliensisinfected-patients with representative polypeptides of the presentinvention in a standard ELISA. Values are expressed as absorbance at 405nm.

[0029]FIGS. 13A and 13B illustrate the level of secreted IL-4 and IFN-γ(in pg/mL) stimulated in mouse lymph node cultures by the addition ofrepresentative polypeptides of the present invention.

[0030]FIG. 14 shows the level of IFN-γ (in pg/mL) secreted byLeishmania-infected and uninfected human PBMC stimulated by theLeishmania antigen M15, as compared to the levels stimulated by L. majorlysate and L-Rack, an antigen that does not appear to be recognized byLeishmania-infected humans.

[0031]FIG. 15 shows the level of IFN-γ (in pg/mL) secreted by infectedand uninfected human PBMC stimulated by soluble Leishmania antigens (Santigens), as compared to the levels stimulated by L. major lysate andL-Rack.

[0032]FIG. 16 illustrates the proliferation of murine lymph nodecultures stimulated by the addition of representative polypeptides ofthe present invention. Values are expressed as cpm.

[0033]FIG. 17 shows the proliferation of human PBMC, prepared fromLeishmania-immune and uninfected individuals, stimulated by M15 ascompared to the proliferation stimulated by L. major lysate and L-Rack.Values are expressed as cpm.

[0034]FIG. 18 illustrates the proliferation of human PBMC, prepared fromLeishmania-infected and uninfected individuals, stimulated by solubleLeishmania antigens as compared to the proliferation stimulated byculture medium, L. major lysate and L-Rack. Values are expressed as cpm.

[0035]FIG. 19 presents a comparison of a Lbhsp83 sequence (SEQ ID NO:6)with homologous sequences from L. amazonensis (Lahsp83) (SEQ ID NO:16),T. cruzi (Tchsp83) (SEQ ID NO:17) and humans (Huhsp89) (SEQ ID NO:18).

[0036]FIG. 20 illustrates the reactivity of rabbit sera raised againstsoluble Leishmania antigens with Leishmania promastigote lysate (lane 1)and soluble Leishmania antigens (lane 2).

[0037]FIG. 21 shows the cDNA and predicted amino acid sequence for theLeishmania antigen Lmsp1a.

[0038]FIG. 22 shows a Southern blot of genomic DNA from L. majordigested with a panel of restriction enzymes (lanes 1 to 7) and sixother Leishmania species digested with PstI (lanes 8 to 13) probed withthe full-length cDNA insert of Lmspla.

[0039]FIG. 23 shows a Southern blot of genomic DNA from L. majordigested with a panel of restriction enzymes, six other Leishmaniaspecies digested with PstI and the infectious pathogens T. cruzi and T.brucei, probed with the full-length cDNA insert of the Leishmaniaantigen MAPS-1A.

[0040]FIG. 24 illustrates the proliferation of PBMC isolated fromuninfected-individuals, patients with active mucosal leishmaniasis andpatients post kala-azar infection, stimulated by MAPS-1A.

[0041]FIG. 25 illustrates the proliferation of murine lymph nodecultures stimulated by MAPS-1A.

[0042]FIG. 26 illustrates the reactivity of MAPS-1A with sera from humanleishmaniasis patients.

[0043]FIG. 27 illustrates the reactivity of MAPS-1A with sera from miceimmunized against and/or infected with leishmaniasis.

[0044]FIG. 28 illustrates the effectiveness of immunization with eithersoluble Leishmania antigens or a mixture of Ldp23, LbeiF4A and M15 plusadjuvant in conferring protection against infection (as measured byfootpad swelling) in a murine leishmaniasis model system, as compared tothe administration of adjuvant alone.

[0045]FIG. 29 illustrates the effectiveness of immunization with MAPS-1Aplus adjuvant in conferring protection against infection (as measured byfootpad swelling) in a murine leishmaniasis model system, as compared tothe administration of adjuvant alone.

[0046]FIGS. 30A and B illustrate the proliferation of murine lymph nodecultures stimulated with either LcgSP8, LcgSP10 or LcgSP3.

[0047]FIG. 31 illustrates the effectiveness of immunization with solubleLeishmania antigens, MAPS-1A and M15 plus adjuvant, IL-12, in conferringprotection against infection (as measured by footpad swelling) in amurine leishmaniasis model system, as compared to the administration ofadjuvant IL-12 alone.

[0048]FIG. 32 illustrates the effectiveness of immunization with M15 DNAand MAPS-1A DNA in conferring protection against infection (as measuredby footpad swelling) in a murine leishmaniasis model system, as comparedto control DNA and saline.

[0049]FIG. 33 illustrates the effectiveness of immunization withLeishmania fusion proteins plus IL-12 as adjuvant, in conferringprotection against infection in a murine leishmaniasis model system.

[0050]FIG. 34 illustrates the effectiveness of immunization withLeishmania fusion proteins plus the adjuvant MPL-SE, in conferringprotection against infection in a murine leishmaniasis model system.

DETAILED DESCRIPTION OF THE INVENTION

[0051] As noted above, the present invention is generally directed tocompositions and methods for preventing, treating and detectingleishmaniasis, as well as for stimulating immune responses in patients.The compositions of the subject invention include polypeptides thatcomprise at least an immunogenic portion of a Leishmania antigen, or avariant of such an antigen. In one preferred embodiment, compositions ofthe present invention include multiple polypeptides selected so as toprovide enhanced protection against a variety of Leishmania species.

[0052] Polypeptides within the scope of the present invention include,but are not limited to, polypeptides comprising immunogenic portions ofLeishmania antigens comprising the sequences recited in SEQ ID NO:2(referred to herein as M15), SEQ ID NO:4 (referred to herein as Ldp23),SEQ ID NO:6 (referred to herein as Lbhsp83), SEQ ID NO:8 (referred toherein as Lt-210), SEQ ID NO:10 (referred to herein as LbeIF4A), SEQ IDNO: 20 (referred to herein as Lmspla), SEQ ID NO: 22 (referred to hereinas Lmsp9a), SEQ ID NOs: 24 and 26 (referred to herein as MAPS-1A), andSEQ ID NO: 36-38, 41, 50-53, 82, 104, 106, 108, 110, 112 and 118-122. Asused herein, the term “polypeptide” encompasses amino acid chains of anylength, including full length proteins (i.e., antigens), wherein theamino acid residues are linked by covalent bonds. Thus, a polypeptidecomprising an immunogenic portion of one of the above antigens mayconsist entirely of the immunogenic portion, or may contain additionalsequences. The additional sequences may be derived from the nativeLeishmania antigen or may be heterologous, and such sequences may (butneed not) be immunogenic. An antigen “having” a particular sequence isan antigen that contains, within its full length sequence, the recitedsequence. The native antigen may, or may not, contain additional aminoacid sequence.

[0053] An immunogenic portion of a Leishmania antigen is a portion thatis capable of eliciting an immune response (i.e., cellular and/orhumoral) in a presently or previously Leishmania-infected patient (suchas a human or a dog) and/or in cultures of lymph node cells orperipheral blood mononuclear cells (PBMC) isolated from presently orpreviously Leishmania-infected individuals. The cells in which aresponse is elicited may comprise a mixture of cell types or may containisolated component cells (including, but not limited to, T-cells, NKcells, macrophages, monocytes and/or B cells). In particular,immunogenic portions are capable of inducing T-cell proliferation and/ora dominantly Th1-type cytokine response (e.g., IL-2, IFN-γ, and/or TNF-αproduction by T-cells and/or NK cells; and/or IL-12 production bymonocytes, macrophages and/or B cells). Immunogenic portions of theantigens described herein may generally be identified using techniquesknown to those of ordinary skill in the art, including therepresentative methods provided herein.

[0054] The compositions and methods of the present invention alsoencompass variants of the above polypeptides. A polypeptide “variant,”as used herein, is a polypeptide that differs from a native protein inone or more substitutions, deletions, additions and/or insertions, suchthat the immunogenicity of the polypeptide is not substantiallydiminished. In other words, the ability of a variant to react withantigen-specific antisera may be enhanced or unchanged, relative to thenative protein, or may be diminished by less than 50%, and preferablyless than 20%, relative to the native protein. Such variants maygenerally be identified by modifying one of the above polypeptidesequences and evaluating the reactivity of the modified polypeptide withantigen-specific antibodies or antisera as described herein. Preferredvariants include those in which one or more portions, such as anN-terminal leader sequence or transmembrane domain, have been removed.Other preferred variants include variants in which a small portion(e.g., 1-30 amino acids, preferably 5-15 amino acids) has been removedfrom the N- and/or C-terminal of the mature protein.

[0055] Polypeptide variants encompassed by the present invention includethose exhibiting at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more identity (determined as describedbelow) to the polypeptides disclosed herein.

[0056] Preferably, a variant contains conservative substitutions. A“conservative substitution” is one in which an amino acid is substitutedfor another amino acid that has similar properties, such that oneskilled in the art of peptide chemistry would expect the secondarystructure and hydropathic nature of the polypeptide to be substantiallyunchanged. Amino acid substitutions may generally be made on the basisof similarity in polarity, charge, solubility, hydrophobicity,hydrophilicity and/or the amphipathic nature of the residues. Forexample, negatively charged amino acids include aspartic acid andglutamic acid; positively charged amino acids include lysine andarginine; and amino acids with uncharged polar head groups havingsimilar hydrophilicity values include leucine, isoleucine and valine;glycine and alanine; asparagine and glutamine; and serine, threonine,phenylalanine and tyrosine. Other groups of amino acids that mayrepresent conservative changes include: (1) ala, pro, gly, glu, asp,gln, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala,phe; (4) lys, arg, his; and (5) phe, tyr, trp, his. A variant may also,or alternatively, contain nonconservative changes. In a preferredembodiment, variant polypeptides differ from a native sequence bysubstitution, deletion or addition of five amino acids or fewer.Variants may also (or alternatively) be modified by, for example, thedeletion or addition of amino acids that have minimal influence on theimmunogenicity, secondary structure and hydropathic nature of thepolypeptide.

[0057] Polynucleotides may comprise a native sequence (i.e., anendogenous sequence that encodes a protein or a portion thereof) or maycomprise a variant, or a biological or antigenic functional equivalentof such a sequence. Polynucleotide variants may contain one or moresubstitutions, additions, deletions and/or insertions, as furtherdescribed below, preferably such that the immunogenicity of the encodedpolypeptide is not diminished, relative to a native tumor protein. Theeffect on the immunogenicity of the encoded polypeptide may generally beassessed as described herein. The term “variants” also encompasseshomologous genes of xenogenic origin.

[0058] When comparing polynucleotide or polypeptide sequences, twosequences are said to be “identical” if the sequence of nucleotides oramino acids in the two sequences is the same when aligned for maximumcorrespondence, as described below. Comparisons between two sequencesare typically performed by comparing the sequences over a comparisonwindow to identify and compare local regions of sequence similarity. A“comparison window” as used herein, refers to a segment of at leastabout 20 contiguous positions, usually 30 to about 75, 40 to about 50,in which a sequence may be compared to a reference sequence of the samenumber of contiguous positions after the two sequences are optimallyaligned.

[0059] Optimal alignment of sequences for comparison may be conductedusing the Megalign program in the Lasergene suite of bioinformaticssoftware (DNASTAR, Inc., Madison, Wis.), using default parameters. Thisprogram embodies several alignment schemes described in the followingreferences: Dayhoff, M. O. (1978) A model of evolutionary change inproteins—Matrices for detecting distant relationships. In Dayhoff, M. O.(ed.) Atlas of Protein Sequence and Structure, National BiomedicalResearch Foundation, Washington D.C. Vol. 5, Suppl. 3, pp. 345-358; HeinJ. (1990) Unified Approach to Alignment and Phylogenes pp. 626-645Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, Calif.;Higgins, D. G. and Sharp, P. M. (1989) CABIOS 5:151-153; Myers, E. W.and Muller W. (1988) CABIOS 4:11-17; Robinson, E. D. (1971) Comb. Theor11:105; Santou, N. Nes, M. (1987) Mol. Biol. Evol. 4:406-425; Sneath, P.H. A. and Sokal, R. R. (1973) Numerical Taxonomy—the Principles andPractice of Numerical Taxonomy, Freeman Press, San Francisco, Calif.;Wilbur, W. J. and Lipman, D. J. (1983) Proc. Natl. Acad., Sci. USA80:726-730.

[0060] Alternatively, optimal alignment of sequences for comparison maybe conducted by the local identity algorithm of Smith and Waterman(1981) Add. APL. Math 2:482, by the identity alignment algorithm ofNeedleman and Wunsch (1970) J. Mol. Biol. 48:443, by the search forsimilarity methods of Pearson and Lipman (1988) Proc. Natl. Acad. Sci.USA 85: 2444, by computerized implementations of these algorithms (GAP,BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics SoftwarePackage, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis.),or by inspection.

[0061] One preferred example of algorithms that are suitable fordetermining percent sequence identity and sequence similarity are theBLAST and BLAST 2.0 algorithms, which are described in Altschul et al.(1977) Nucl. Acids Res. 25:3389-3402 and Altschul et al. (1990) J. Mol.Biol. 215:403-410, respectively. BLAST and BLAST 2.0 can be used, forexample with the parameters described herein, to determine percentsequence identity for the polynucleotides and polypeptides of theinvention. Software for performing BLAST analyses is publicly availablethrough the National Center for Biotechnology Information. In oneillustrative example, cumulative scores can be calculated using, fornucleotide sequences, the parameters M (reward score for a pair ofmatching residues; always >0) and N (penalty score for mismatchingresidues; always <0). For amino acid sequences, a scoring matrix can beused to calculate the cumulative score. Extension of the word hits ineach direction are halted when: the cumulative alignment score falls offby the quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, andexpectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff andHenikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915) alignments, (B) of50, expectation (E) of 10, M=5, N=-4 and a comparison of both strands.

[0062] Preferably, the “percentage of sequence identity” is determinedby comparing two optimally aligned sequences over a window of comparisonof at least 20 positions, wherein the portion of the polynucleotide orpolypeptide sequence in the comparison window may comprise additions ordeletions (i.e., gaps) of 20 percent or less, usually 5 to 15 percent,or 10 to 12 percent, as compared to the reference sequences (which doesnot comprise additions or deletions) for optimal alignment of the twosequences. The percentage is calculated by determining the number ofpositions at which the identical nucleic acid bases or amino acidresidue occurs in both sequences to yield the number of matchedpositions, dividing the number of matched positions by the total numberof positions in the reference sequence (i.e., the window size) andmultiplying the results by 100 to yield the percentage of sequenceidentity.

[0063] Therefore, the present invention encompasses polynucleotide andpolypeptide sequences having substantial identity to the sequencesdisclosed herein, for example those comprising at least 50% sequenceidentity, preferably at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 96%, 97%, 98%, or 99% or higher, sequence identity compared to apolynucleotide or polypeptide sequence of this invention using themethods described herein, (e.g., BLAST analysis using standardparameters, as described below). One skilled in this art will recognizethat these values can be appropriately adjusted to determinecorresponding identity of proteins encoded by two nucleotide sequencesby taking into account codon degeneracy, amino acid similarity, readingframe positioning and the like.

[0064] In additional embodiments, the present invention providesisolated polynucleotides and polypeptides comprising various lengths ofcontiguous stretches of sequence identical to or complementary to one ormore of the sequences disclosed herein. For example, polynucleotides areprovided by this invention that comprise at least about 15, 20, 30, 40,50, 75, 100, 150, 200, 300, 400, 500 or 1000 or more contiguousnucleotides of one or more of the sequences disclosed herein as well asall intermediate lengths there between. It will be readily understoodthat “intermediate lengths”, in this context, means any length betweenthe quoted values, such as 16, 17, 18, 19, etc.; 21, 22, 23, etc.; 30,31, 32, etc.; 50, 51, 52, 53, etc.; 100, 101, 102, 103, etc.; 150, 151,152, 153, etc.; including all integers through 200-500; 500-1,000, andthe like.

[0065] The polynucleotides of the present invention, or fragmentsthereof, regardless of the length of the coding sequence itself, may becombined with other DNA sequences, such as promoters, polyadenylationsignals, additional restriction enzyme sites, multiple cloning sites,other coding segments, and the like, such that their overall length mayvary considerably. It is therefore contemplated that a nucleic acidfragment of almost any length may be employed, with the total lengthpreferably being limited by the ease of preparation and use in theintended recombinant DNA protocol. For example, illustrative DNAsegments with total lengths of about 10,000, about 5000, about 3000,about 2,000, about 1,000, about 500, about 200, about 100, about 50 basepairs in length, and the like, (including all intermediate lengths) arecontemplated to be useful in many implementations of this invention.

[0066] In other embodiments, the present invention is directed topolynucleotides that are capable of hybridizing under moderatelystringent conditions to a polynucleotide sequence provided herein, or afragment thereof, or a complementary sequence thereof. Hybridizationtechniques are well known in the art of molecular biology. For purposesof illustration, suitable moderately stringent conditions for testingthe hybridization of a polynucleotide of this invention with otherpolynucleotides include prewashing in a solution of 5× SSC, 0.5% SDS,1.0 mM EDTA (pH 8.0); hybridizing at 50° C-65° C., 5× SSC, overnight;followed by washing twice at 65° C. for 20 minutes with each of 2×, 0.5×and 0.2× SSC containing 0.1% SDS.

[0067] Moreover, it will be appreciated by those of ordinary skill inthe art that, as a result of the degeneracy of the genetic code, thereare many nucleotide sequences that encode a polypeptide as describedherein. Some of these polynucleotides bear minimal homology to thenucleotide sequence of any native gene. Nonetheless, polynucleotidesthat vary due to differences in codon usage are specificallycontemplated by the present invention. Further, alleles of the genescomprising the polynucleotide sequences provided herein are within thescope of the present invention. Alleles are endogenous genes that arealtered as a result of one or more mutations, such as deletions,additions and/or substitutions of nucleotides. The resulting mRNA andprotein may, but need not, have an altered structure or function.Alleles may be identified using standard techniques (such ashybridization, amplification and/or database sequence comparison).

[0068] “Polypeptides” as described herein also include combinationpolypeptides, also referred to as fusion proteins. A “combinationpolypeptide” is a polypeptide comprising at least one of the aboveimmunogenic portions and one or more additional immunogenic Leishmaniasequences, which are joined via a peptide linkage into a single aminoacid chain. The sequences may be joined directly (i.e., with nointervening amino acids) or may be joined by way of a linker sequence(e.g., Gly-Cys-Gly) that does not significantly diminish the immunogenicproperties of the component polypeptides.

[0069] Fusion proteins may generally be prepared using standardtechniques, including chemical conjugation. Preferably, a fusion proteinis expressed as a recombinant protein, allowing the production ofincreased levels, relative to a non-fused protein, in an expressionsystem. Briefly, DNA sequences encoding the polypeptide components maybe assembled separately, and ligated into an appropriate expressionvector. The 3′ end of the DNA sequence encoding one polypeptidecomponent is ligated, with or without a peptide linker, to the 5′ end ofa DNA sequence encoding the second polypeptide component so that thereading frames of the sequences are in frame. This permits translationinto a single fusion protein that retains the biological activity ofboth component polypeptides.

[0070] A peptide linker sequence may be employed to separate the firstand the second polypeptide components by a distance sufficient to ensurethat each polypeptide folds into its secondary and tertiary structures.Such a peptide linker sequence is incorporated into the fusion proteinusing standard techniques well known in the art. Suitable peptide linkersequences may be chosen based on the following factors: (1) theirability to adopt a flexible extended conformation; (2) their inabilityto adopt a secondary structure that could interact with functionalepitopes on the first and second polypeptides; and (3) the lack ofhydrophobic or charged residues that might react with the polypeptidefunctional epitopes. Preferred peptide linker sequences contain Gly, Asnand Ser residues. Other near neutral amino acids, such as Thr and Alamay also be used in the linker sequence. Amino acid sequences which maybe usefully employed as linkers include those disclosed in Maratea etal., Gene 40:39-46, 1985; Murphy et al., Proc. Natl. Acad Sci. USA83:8258-8262, 1986; U.S. Pat. Nos. 4,935,233 and 4,751,180. The linkersequence may generally be from 1 to about 50 amino acids in length.Linker sequences are not required when the first and second polypeptideshave non-essential N-terminal amino acid regions that can be used toseparate the functional domains and prevent steric interference.

[0071] The ligated DNA sequences are operably linked to suitabletranscriptional or translational regulatory elements. The regulatoryelements responsible for expression of DNA are located only 5′ to theDNA sequence encoding the first polypeptides. Similarly, stop codonsrequired to end translation and transcription termination signals areonly present 3′ to the DNA sequence encoding the second polypeptide. Thepreparation of fusion proteins of Leishmania antigens is described indetail below in Example 19.

[0072] In general, Leishmania antigens having immunogenic properties,and DNA sequences encoding such antigens, may be prepared using any of avariety of procedures from one or more Leishmania species including, butnot limited to, L. donovani, L. chagasi, L. infantum, L. major, L.amazonensis, L. braziliensis, L. panamensis, L. mexicana, L. tropica,and L. guyanensis. Such species are available, for example, from theAmerican Type Culture Collection (ATCC), Rockville, Md. For example,peptides isolated from MHC class II molecules of macrophages infectedwith a Leishmania species may be used to rescue the correspondingLeishmania donor antigens. MHC class II molecules are expressed mainlyby cells of the immune system, including macrophages. These moleculespresent peptides, which are usually 13-17 amino acids long, derived fromforeign antigens that are degraded in cellular vesicles. The boundpeptide antigens are then recognized by CD4 T-cells. Accordingly,foreign peptides isolated from MHC class II molecules of, for example,Leishmania-infected murine macrophages may be used to identifyimmunogenic Leishmania proteins.

[0073] Briefly, peptides derived from Leishmania antigens may beisolated by comparing the reverse phase HPLC profile of peptidesextracted from infected macrophages with the profile of peptidesextracted from uninfected cells. Peptides giving rise to distinct HPLCpeaks unique to infected macrophages may then be sequenced using, forexample, Edman chemistry as described in Edman and Berg, Eur J Biochem,80:116-132 (1967). A DNA fragment corresponding to a portion of aLeishmania gene encoding the peptide may then be amplified from aLeishmania cDNA library using an oligonucleotide sense primer derivedfrom the peptide sequence and an oligo dT antisense primer. Theresulting DNA fragment may then be used as a probe to screen aLeishmania library for a full length cDNA or genomic clone that encodesthe Leishmania antigen. Such screens may generally be performed usingtechniques well known to those of ordinary skill in the art, such asthose described in Sambrook et al., Molecular Cloning: 4 LaboratoryManual, Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y.(1989).

[0074] This approach may be used to identify a 23 kD Leishmania donovaniantigen (referred to herein as Ldp23). The sequence of a polynucleotideencoding Ldp23 is provided in SEQ ID NO:3 and the amino acid sequence ofLdp23 is provided in SEQ ID NO:4. Using the methods described herein,Ldp23 has been shown to induce a Th1 immune response in T-cells preparedfrom Leishmania-infected mice.

[0075] Alternatively, a Leishmania cDNA or genomic expression librarymay be screened with serum from a Leishmania-infected individual, usingtechniques well known to those of ordinary skill in the art.Polynucleotides encoding reactive antigens may then be used to expressthe recombinant antigen for purification. The immunogenic properties ofthe purified Leishmania antigens may then be evaluated using, forexample the representative methods described herein.

[0076] For example, sera from Leishmania-infected mice may be used toscreen a cDNA library prepared from Leishmania amastigotes. Reactiveclones may then be expressed and recombinant proteins assayed for theability to stimulate T-cells or NK cells derived from Leishmania-immuneindividuals (i.e., individuals having evidence of infection, asdocumented by positive serological reactivity with Leishmania-specificantibodies and/or a Leishmania-specific DTH response, without clinicalsymptoms of leishmaniasis). This procedure may be used to obtain arecombinant polynucleotide encoding the Leishmania antigen designatedM15. The sequence of such a polynucleotide is provided in SEQ ID NO:1,and the amino acid sequence of the encoded protein is provided in SEQ IDNO:2.

[0077] A similar approach may be used to isolate a genomicpolynucleotide encoding an immunogenic Leishmania braziliensis antigen,referred to herein as Lbhsp83. More specifically, a genomic cloneencoding Lbhsp83 may be isolated by screening a L. braziliensisexpression library with sera from a Leishmania-infected individual. TheDNA encoding Lbhsp83 is homologous to the gene encoding the eukaryotic83 kD heat shock protein. The sequence of a polynucleotide encodingnearly all of Lbhsp83 is presented in SEQ ID NO:5, and the encoded aminoacid sequence is provided in SEQ ID NO:6. Using the methods describedbelow, Lbhsp83 has been found to stimulate proliferation, and a mixedTh1 and Th2 cytokine profile, in PBMC isolated from L.braziliensis-infected patients. Accordingly, Lbhsp83 is an immunogenicLeishmania antigen. Regions of Lbhsp83 that are not conserved with themammalian gene have been found to be particularly potent for T-cellstimulation and antibody binding. Such regions may be identified, forexample, by visual inspection of the sequence comparison provided inFIG. 19.

[0078] This approach may also be used to isolate a polynucleotideencoding a 210 kD immunogenic L. tropica antigen, referred to herein asLt-210. The preparation and characterization of Lt-210, and immunogenicportions thereof (such as Lt-1 and immunogenic repeat and non-repeatsequences), is described in detail in U.S. patent application Ser. No.08/511,872, filed Aug. 4, 1995. The sequence of a polynucleotideencoding Lt-1 is provided in SEQ ID NO:7 and the encoded amino acidsequence is presented in SEQ ID NO:8.

[0079] The above approach may further be used to isolate apolynucleotide encoding a L. braziliensis antigen referred to herein asLbeIF4A. Briefly, such a clone may be isolated by screening a L.braziliensis expression library with sera obtained from a patientafflicted with mucosal leishmaniasis, and analyzing the reactiveantigens for the ability to stimulate proliferative responses andpreferential Th1 cytokine production in PBMC isolated fromLeishmania-infected patients, as described below. The preparation andcharacterization of LbeIF4A is described in detail in U.S. patentapplication Ser. Nos. 08/454,036 and 08/488,386, which arecontinuations-in-part of U.S. patent application Ser. No. 08/232,534,filed Apr. 22, 1994. The sequence of a polynucleotide encoding LbeIF4Ais provided in SEQ ID NO:9 and the encoded amino acid sequence ispresented in SEQ ID NO:10. Homologs of LbeIF4A, such as that found in L.major, may also be isolated using this approach, and are within thescope of the present invention.

[0080] Compositions of the present invention may also, or alternatively,contain soluble Leishmania antigens. As used herein, “soluble Leishmaniaantigens” refers to a mixture of at least 8 different Leishmaniaantigens that may be isolated from the supernatant of Leishmaniapromastigotes of any species grown for 8-12 hours in protein-freemedium. Briefly, the organisms are grown to late log phase in complexmedium with serum until they reach a density of 2-3×10⁷ viable organismsper mL of medium. The organisms are thoroughly washed to remove mediumcomponents and resuspended at 2-3×10⁷ viable organisms per mL of definedserum-free medium consisting of equal parts RPMI 1640 and medium 199,both from Gibco BRL, Gaithersburg, Md. After 8-12 hours, the supernatantcontaining soluble Leishmania antigens is removed, concentrated 10 foldand dialyzed against phosphate-buffered saline for 24 hours. Thepresence of at least eight different antigens within the mixture ofLeishmania antigens may be confirmed using SDS-PAGE (i.e., through theobservation of at least 8 different bands). The immunogenic propertiesof the soluble Leishmania antigens may be confirmed by evaluating theability of the preparation to elicit an immune response in cultures oflymph node cells and/or peripheral blood mononuclear cells (PBMC)isolated from presently or previously Leishmania-infected individuals.Such an evaluation may be performed as described below.

[0081] Individual antigens present within the mixture of solubleLeishmania antigens may be isolated by immunizing mice or rabbits withLeishmania culture supernatant, containing soluble antigens, andemploying the resultant sera to screen a Leishmania cDNA expressionlibrary as described in detail below. This procedure may be used toisolate recombinant polynucleotides encoding the L. major antigensreferred to herein as Lmsp1a, Lmsp9a and MAPS-1A. DNA sequences encodingLmsp1a, Lmsp9a and MAPS-1A are provided in SEQ ID NO: 19, 21 and 23,respectively, with the corresponding predicted amino acid sequencesbeing presented in SEQ ID NO: 20, 22 and 24, respectively. Similarly,sera from mice or rabbits immunized with L. major culture supernatantmay be used to screen an L. major genomic DNA library. As detailedbelow, this procedure may be used to isolate polynucleotides encodingthe L. major antigens referred to herein as LmgSP1, LmgSP3, LmgSP5,LmgSP8, LmgSP9, LmgSP13, LmgSP19, and polynucleotides encoding the L.chagasi antigens LcgSP1, LcgSP3, LcgSP4, LcgSP8, and LcgSP10. The DNAsequences encoding these antigens are provided in SEQ ID NO:29-35 and44-48, respectively, with the corresponding amino acid sequences beingprovided in SEQ ID NO: 36-42 and 49-53. The L. major antigens referredto herein as 1G6-34, 1E6-44, 4A5-63, 1B11-39, 2A10-37, 4G2-83, 4H6-41and 8G3-100 may be isolated by means of CD4+ T cell expression cloningas described below. DNA sequences encoding these antigens are providedin SEQ ID NO: 72-79, respectively, with the corresponding predictedamino acid sequences being provided in SEQ ID NO: 80-87. The immunogenicproperties of the isolated Leishmania antigens may be evaluated using,for example, the representative methods described herein.

[0082] Regardless of the method of preparation, the antigens describedherein are immunogenic. In other words, the antigens (and immunogenicportions thereof) are capable of eliciting an immune response incultures of lymph node cells and/or peripheral blood mononuclear cells(PBMC) isolated from presently or previously Leishmania- infectedindividuals. More specifically, the antigens, and immunogenic portionsthereof, have the ability to induce T-cell proliferation and/or toelicit a dominantly Th1-type cytokine response (e.g., IL-2, IFN-γ,and/or TNF-α production by T-cells and/or NK cells; and/or IL-12production by monocytes, macrophages and/or B cells) in cells isolatedfrom presently or previously Leishmania-infected individuals. ALeishmania-infected individual may be afflicted with a form ofleishmaniasis (such as subclinical, cutaneous, mucosal or activevisceral) or may be asymptomatic. Such individuals may be identifiedusing methods known to those of ordinary skill in the art. Individualswith leishmaniasis may be identified based on clinical findingsassociated with at least one of the following: isolation of parasitefrom lesions, a positive skin test with Leishmania lysate or a positiveserological test. Asymptomatic individuals are infected individuals whohave no signs or symptoms of the disease. Such individuals can beidentified based on a positive serological test and/or skin test withLeishmania lysate.

[0083] The term “PBMC,” which refers to a preparation of nucleated cellsconsisting primarily of lymphocytes and monocytes that are present inperipheral blood, encompasses both mixtures of cells and preparations ofone or more purified cell types. PBMC may be isolated by methods knownto those in the art. For example, PBMC may be isolated by densitycentrifugation through, for example, Ficoll™ (Winthrop Laboratories, NewYork). Lymph node cultures may generally be prepared by immunizingBALB/c mice (e.g., in the rear foot pad) with Leishmania promastigotesemulsified in complete Freünd's adjuvant. The draining lymph nodes maybe excised following immunization and T-cells may be purified in ananti-mouse Ig column to remove the B cells, followed by a passagethrough a Sephadex G10 column to remove the macrophages. Similarly,lymph node cells may be isolated from a human following biopsy orsurgical removal of a lymph node.

[0084] The ability of a polypeptide (e.g., a Leishmania antigen or aportion or other variant thereof) to induce a response in PBMC or lymphnode cell cultures may be evaluated by contacting the cells with thepolypeptide and measuring a suitable response. In general, the amount ofpolypeptide that is sufficient for the evaluation of about 2×10⁵ cellsranges from about 10 ng to about 100 μg, and preferably is about 1-10μg. The incubation of polypeptide with cells is typically performed at37° C. for about 1-3 days. Following incubation with polypeptide, thecells are assayed for an appropriate response. If the response is aproliferative response, any of a variety of techniques well known tothose of ordinary skill in the art may be employed. For example, thecells may be exposed to a pulse of radioactive thymidine and theincorporation of label into cellular DNA measured. In general, apolypeptide that results in at least a three fold increase inproliferation above background (i.e., the proliferation observed forcells cultured without polypeptide) is considered to be able to induceproliferation.

[0085] Alternatively, the response to be measured may be the secretionof one or more cytokines (such as interferon-γ (IFN-γ), interleukin-4(IL-4), interleukin-12 (p70 and/or p40), interleukin-2 (IL-2) and/ortumor necrosis factor-α (TNF-α) or the change in the level of mRNAencoding one or more specific cytokines. In particular, the secretion ofinterferon-γ, interleukin-2, tumor necrosis factor-α and/orinterleukin-12 is indicative of a Th1 response, which is responsible forthe protective effect against Leishmania. Assays for any of the abovecytokines may generally be performed using methods known to those ofordinary skill in the art, such as an enzyme-linked immunosorbent assay(ELISA). Suitable antibodies for use in such assays may be obtained froma variety of sources such as Chemicon, Temucula, Calif. and PharMingen,San Diego, Calif., and may generally be used according to themanufacturer's instructions. The level of mRNA encoding one or morespecific cytokines may be evaluated by, for example, amplification bypolymerase chain reaction (PCR). In general, a polypeptide that is ableto induce, in a preparation of about 1-3×10⁵ cells, the production of 30pg/mL of IL-12, IL-4, IFN-γ, TNF-α or IL-12 p40, or 10 pg/mL of IL-12p70, is considered able to stimulate production of a cytokine.

[0086] Immunogenic portions of the antigens described herein may beprepared and identified using well known techniques, such as thosesummarized in Paul, Fundamental Immunology, 3rd ed., 243-247 (RavenPress, 1993) and references cited therein. Such techniques includescreening polypeptides derived from the native antigen for immunogenicproperties using, for example, the representative techniques describedherein. An immunogenic portion of a polypeptide is a portion that,within such representative assays, generates an immune response (e.g.,proliferation and/or cytokine production) that is substantially similarto that generated by the full length antigen. In other words, animmunogenic portion of an antigen may generate at least about 25%, andpreferably at least about 50%, of the response generated by the fulllength antigen in the model ,assays described herein.

[0087] Portions and other variants of immunogenic Leishmania antigensmay be generated by synthetic or recombinant means. Syntheticpolypeptides having fewer than about 100 amino acids, and generallyfewer than about 50 amino acids, may be generated using techniques wellknown to those of ordinary skill in the art. For example, suchpolypeptides may be synthesized using any of the commercially availablesolid-phase techniques, such as the Merrifield solid-phase synthesismethod, where amino acids are sequentially added to a growing amino acidchain. See Merrifield, J. Am. Chem. Soc. 85:2149-2146, 1963. Equipmentfor automated synthesis of polypeptides is commercially available fromsuppliers such as Perkin Elmer/Applied BioSystemsDivision, Foster City,Calif., and may be operated according to the manufacturer'sinstructions.

[0088] Recombinant polypeptides containing portions and/or variants of anative antigen may be readily prepared from a DNA sequence encoding theantigen. For example, supernatants from suitable host/vector systemswhich secrete recombinant protein into culture media may be firstconcentrated using a commercially available filter. Followingconcentration, the concentrate may be applied to a suitable purificationmatrix such as an affinity matrix or an ion exchange resin. Finally, oneor more reverse phase HPLC steps can be employed to further purify arecombinant protein.

[0089] In general, any of a variety of expression vectors known to thoseof ordinary skill in the art may be employed to express recombinantpolypeptides of this invention. Expression may be achieved in anyappropriate host cell that has been transformed or transfected with anexpression vector containing a polynucleotide that encodes a recombinantpolypeptide. Suitable host cells include prokaryotes, yeast and highereukaryotic cells. Preferably, the host cells employed are E. coli, yeastor a mammalian cell line such as COS or CHO. The DNA sequences expressedin this manner may encode naturally occurring antigens, portions ofnaturally occurring antigens, or other variants thereof. For example,variants of a native antigen may generally be prepared using standardmutagenesis techniques, such as oligonucleotide-directed site-specificmutagenesis, and sections of the DNA sequence may be removed to permitpreparation of truncated polypeptides.

[0090] In another aspect, the present invention provides epitope repeatsequences, or antigenic epitopes, of a Leishmania antigen, together withpolypeptides comprising at least two such contiguous antigenic epitopes.As used herein an “epitope” is a portion of an antigen that reacts withsera from Leishmania-infected individuals (i.e. an epitope isspecifically bound by one or more antibodies present in such sera). Asdiscussed above, epitopes of the antigens described in the presentapplication may be generally identified using techniques well known tothose of skill in the art.

[0091] In one embodiment, antigenic epitopes of the present inventioncomprise an amino acid sequence provided in SEQ ID NO:43, 56, 57 or 58.As discussed in more detail below, antigenic epitopes provided hereinmay be employed in the diagnosis and treatment of Leishmania infection,either alone or in combination with other Leishmania antigens orantigenic epitopes. Antigenic epitopes and polypeptides comprising suchepitopes may be prepared by synthetic means, as described generallyabove and in detail in Example 15.

[0092] In certain aspects of the present invention, described in detailbelow, the polypeptides, antigenic epitopes, fusion proteins and/orsoluble Leishmania antigens of the present invention may be incorporatedinto pharmaceutical compositions or vaccines. For clarity, the term“polypeptide” will be used when describing specific embodiments of theinventive therapeutic compositions and diagnostic methods. However, itwill be clear to one of skill in the art that the antigenic epitopes andfusion proteins of the present invention may also be employed in suchcompositions and methods.

[0093] Pharmaceutical compositions comprise one or more polypeptides,each of which may contain one or more of the above sequences (orvariants thereof), and a physiologically acceptable carrier. Vaccines,also referred to as immunogenic compositions, comprise one or more ofthe above polypeptides and an immunostimulant, such as an adjuvant(e.g., LbeIF4A, interleukin-12 or other cytokines) or a liposome (intowhich the polypeptide is incorporated). Many adjuvants contain asubstance designed to protect the antigen from rapid catabolism, such asaluminum hydroxide or mineral oil, and a stimulator of immune responses,such as lipid A, Bortadella pertussis or Mycobacterium tuberculosisderived proteins. Certain adjuvants are commercially available as, forexample, Freund's Incomplete Adjuvant and Complete Adjuvant (DifcoLaboratories, Detroit, Mich.); Merck Adjuvant 65 (Merck and Company,Inc., Rahway, N.J.); AS-2 (SmithKline Beecham, Philadelphia, Pa.);aluminum salts such as aluminum hydroxide gel (alum) or aluminumphosphate; salts of calcium, iron or zinc; an insoluble suspension ofacylated tyrosine; acylated sugars; cationically or anionicallyderivatized polysaccharides; polyphosphazenes; biodegradablemicrospheres; monophosphoryl lipid A and quil A. Cytokines, such asGM-CSF, interleukin-2, -7, -12, and other like growth factors, may alsobe used as adjuvants.

[0094] Within certain embodiments of the invention, the adjuvantcomposition is preferably one that induces an immune responsepredominantly of the Th1 type. By virtue of its ability to induce anexclusive Th1 immune response, the use of LbeIF4A, and variants thereof,as an adjuvant in the vaccines of the present invention is particularlypreferred. Certain other preferred adjuvants for eliciting apredominantly Th1 -type response include, for example, a combination ofmonophosphoryl lipid A, preferably 3-de-O-acylated monophosphoryl lipidA, together with an aluminum salt. MPL® adjuvants are available fromCorixa Corporation (Seattle, WA; see, for example, U.S. Pat. Nos.4,436,727; 4,877,611; 4,866,034 and 4,912,094). CpG-containingoligonucleotides (in which the CpG dinucleotide is unmethylated) alsoinduce a predominantly Th1 response. Such oligonucleotides are wellknown and are described, for example, in WO 96/02555, WO 99/33488 andU.S. Pat. Nos. 6,008,200 and 5,856,462. Immunostimulatory DNA sequencesare also described, for example, by Sato et al., Science 273:352, 1996.Another preferred adjuvant comprises a saponin, such as Quil A, orderivatives thereof, including QS21 and QS7 (Aquila BiopharmaceuticalsInc., Framingham, Mass.); Escin; Digitonin; or Gypsophila or Chenopodiumquinoa saponins. Other preferred formulations include more than onesaponin in the adjuvant combinations of the present invention, forexample combinations of at least two of the following group comprisingQS21, QS7, Quil A, β-escin, or digitonin.

[0095] Alternatively the saponin formulations may be combined withvaccine vehicles composed of chitosan or other polycationic polymers,polylactide and polylactide-co-glycolide particles, poly-N-acetylglucosamine-based polymer matrix, particles composed of polysaccharidesor chemically modified polysaccharides, liposomes and lipid-basedparticles, particles composed of glycerol monoesters, etc. The saponinsmay also be formulated in the presence of cholesterol to formparticulate structures such as liposomes or ISCOMs. Furthermore, thesaponins may be formulated together with a polyoxyethylene ether orester, in either a non-particulate solution or suspension, or in aparticulate structure such as a paucilamelar liposome or ISCOM. Thesaponins may also be formulated with excipients such as Carbopol^(R) toincrease viscosity, or may be formulated in a dry powder form with apowder excipient such as lactose.

[0096] In one preferred embodiment, the adjuvant system includes thecombination of a monophosphoryl lipid A and a saponin derivative, suchas the combination of QS21 and 3D-MPL® adjuvant, as described in WO94/00153, or a less reactogenic composition where the QS21 is quenchedwith cholesterol, as described in WO 96/33739. Other preferredformulations comprise an oil-in-water emulsion and tocopherol. Anotherparticularly preferred adjuvant formulation employing QS21, 3D-MPL®adjuvant and tocopherol in an oil-in-water emulsion is described in WO95/17210.

[0097] Another enhanced adjuvant system involves the combination of aCpG-containing oligonucleotide and a saponin derivative particularly thecombination of CpG and QS21 is disclosed in WO 00/09159. Preferably theformulation additionally comprises an oil in water emulsion andtocopherol.

[0098] Additional illustrative adjuvants for use in the compositions ofthe invention include Montanide ISA 720 (Seppic, France), SAF (Chiron,Calif., United States), ISCOMS (CSL), MF-59 (Chiron), the SBAS series ofadjuvants (e.g., SBAS-2 or SBAS-4, available from SmithKline Beecham,Rixensart, Belgium), Enhanzyn™ (Corixa, Hamilton, Mont.), RC-529(Corixa, Hamilton, Mont.) and other aminoalkyl glucosaminide4-phosphates (AGPs), such as those described in U.S. Pat. No. 6,113,918and pending U.S. patent application Ser. No. 09/074,720, the disclosuresof which are incorporated herein by reference in their entireties, andpolyoxyethylene ether adjuvants such as those described in WO99/52549A1.

[0099] Other preferred adjuvants include adjuvant molecules of thegeneral formula

HO(CH₂CH₂O)_(n)—A—R,  (I)

[0100] wherein, n is 1-50, A is a bond or —C(O)—, R is C₁₋₅₀ alkyl orPhenyl C₁₋₅₀ alkyl.

[0101] One embodiment of the present invention consists of a vaccineformulation comprising a polyoxyethylene ether of general formula (I),wherein n is between 1 and 50, preferably 4-24, most preferably 9; the Rcomponent is C₁₋₅₀, preferably C₄-C₂₀ alkyl and most preferably C₁₂alkyl, and A is a bond. The concentration of the polyoxyethylene ethersshould be in the range 0.1-20%, preferably from 0.1-10%, and mostpreferably in the range 0.1-1%. Preferred polyoxyethylene ethers areselected from the following group: polyoxyethylene-9-lauryl ether,polyoxyethylene-9-steoryl ether, polyoxyethylene-8-steoryl ether,polyoxyethylene-4-lauryl ether, polyoxyethylene-35-lauryl ether, andpolyoxyethylene-23-lauryl ether. Polyoxyethylene ethers such aspolyoxyethylene lauryl ether are described in the Merck index (12^(th)edition: entry 7717). These adjuvant molecules are described in WO99/52549. The polyoxyethylene ether according to the general formula (I)above may, if desired, be combined with another adjuvant. For example, apreferred adjuvant combination is preferably with CpG as described inthe pending UK patent application GB 9820956.2.

[0102] Vaccines may additionally contain a delivery vehicle, such as abiodegradable microsphere (disclosed, for example, in U.S. Pat. Nos.4,897,268 and 5,075,109). Pharmaceutical compositions and vaccineswithin the scope of the present invention may also contain otherLeishmania antigens, either incorporated into a combination polypeptideor present within one or more separate polypeptides.

[0103] Alternatively, a pharmaceutical or immunogenic composition maycontain an immunostimulant, such as an adjuvant (e.g., LbeIF4A,interleukin-12 or other cytokines, or DNA coding for such enhancers),and DNA encoding one or more of the polypeptides or fusion proteinsdescribed above, such that the polypeptide is generated in situ. In suchcompositions, the DNA may be present within any of a variety of deliverysystems known to those of ordinary skill in the art, including nucleicacid expression systems, bacteria and viral expression systems.Appropriate nucleic acid expression systems contain the necessary DNAsequences for expression in the patient (such as a suitable promoter andterminating signal). Bacterial delivery systems involve theadministration of a bacterium (such as Bacillus-Calmette-Guerrin) thatexpresses an immunogenic portion of the polypeptide on its cell surface.In a preferred embodiment, the DNA may be introduced using a viralexpression system (e.g., vaccinia or other pox virus, retrovirus, oradenovirus), which may involve the use of a non-pathogenic (defective),replication competent virus. Techniques for incorporating DNA into suchexpression systems are well known to those of ordinary skill in the art.The DNA may also be “naked,” as described, for example, in Ulmer et al.,Science 259:1745-1749 (1993) and reviewed by Cohen, Science259:1691-1692 (1993). The uptake of naked DNA may be increased bycoating the DNA onto biodegradable beads, which are efficientlytransported into the cells.

[0104] While any suitable carrier known to those of ordinary skill inthe art may be employed in the pharmaceutical compositions of thisinvention, the type of carrier will vary depending on the mode ofadministration. For parenteral administration, such as subcutaneousinjection, the carrier preferably comprises water, saline, alcohol, afat, a wax or a buffer. For oral administration, any of the abovecarriers or a solid carrier, such as mannitol, lactose, starch,magnesium stearate, sodium saccharine, talcum, cellulose, glucose,sucrose, and magnesium carbonate, may be employed. Biodegradablemicrospheres (e.g., polylactic galactide) may also be employed ascarriers for the pharmaceutical compositions of this invention. Suitablebiodegradable microspheres are disclosed, for example, in U.S. Pat. Nos.4,897,268 and 5,075,109.

[0105] In one preferred embodiment, compositions of the presentinvention include multiple polypeptides selected so as to provideenhanced protection against a variety of Leishmania species. Suchpolypeptides may be selected based on the species of origin of thenative antigen or based on a high degree of conservation of amino acidsequence among different species of Leishmania. A combination ofindividual polypeptides may be particularly effective as a prophylacticand/or therapeutic vaccine because (1) stimulation of proliferationand/or cytokine production by a combination of individual polypeptidesmay be additive, (2) stimulation of proliferation and/or cytokineproduction by a combination of individual polypeptides may besynergistic, (3) a combination of individual polypeptides may stimulatecytokine profiles in such a way as to be complementary to each otherand/or (4) individual polypeptides may be complementary to one anotherwhen certain of them are expressed more abundantly on the individualspecies or strain of Leishmania responsible for infection. A preferredcombination contains polypeptides that comprise immunogenic portions ofM15, Ldp23, Lbhsp83, Lt-1 and LbeIF4A. Alternatively, or in addition,the combination may include one or more polypeptides comprisingimmunogenic portions of other Leishmania antigens disclosed herein,and/or soluble Leishmania antigens.

[0106] In another preferred embodiment, compositions of the presentinvention include single polypeptides selected so as to provide enhancedprotection against a variety of Leishmania species. A single individualpolypeptide may be particularly effective as a prophylactic and/ortherapeutic vaccine for those reasons stated above for combinations ofindividual polypeptides.

[0107] In another embodiment, compositions of the present inventioninclude individual polypeptides and combinations of the above describedpolypeptides employed with a variety of adjuvants, such as IL-12(protein or DNA) to confer a protective response against a variety ofLeishmania species.

[0108] In yet another embodiment, compositions of the present inventioninclude DNA constructs of the various Leishmania species employed aloneor in combination with variety of adjuvants, such as IL-12 (protein orDNA) to confer a protective response against a variety of Leishmaniaspecies.

[0109] The above pharmaceutical compositions and vaccines may be used,for example, to induce protective immunity against Leishmania in apatient, such as a human or a dog, to prevent leishmaniasis. Appropriatedoses and methods of administration for this purposes are described indetail below.

[0110] The pharmaceutical and immunogenic compositions described hereinmay also be used to stimulate an immune response, which may be cellularand/or humoral, in a patient. For Leishmania-infected patients, theimmune responses that may be generated include a preferential Th1 immuneresponse (i.e., a response characterized by the production of thecytokines interleukin-1, interleukin-2, interleukin-12 and/orinterferon-γ, as well as tumor necrosis factor-α). For uninfectedpatients, the immune response may be the production of interleukin-12and/or interleukin-2, or the stimulation of gamma delta T-cells. Ineither category of patient, the response stimulated may include IL-12production. Such responses may also be elicited in biological samples ofPBMC or components thereof derived from Leishmania-infected oruninfected individuals. As noted above, assays for any of the abovecytokines may generally be performed using methods known to those ofordinary skill in the art, such as an enzyme-linked immunosorbent assay(ELISA).

[0111] Suitable pharmaceutical compositions and vaccines for use in thisaspect of the present invention are those that contain at least onepolypeptide comprising an immunogenic portion of a Leishmania antigendisclosed herein (or a variant thereof). Preferably, the polypeptidesemployed in the pharmaceutical compositions and vaccines arecomplementary, as described above. Soluble Leishmania antigens, with orwithout additional polypeptides, may also be employed.

[0112] The pharmaceutical compositions and vaccines described herein mayalso be used to treat a patient afflicted with a disease responsive toIL-12 stimulation. The patient may be any warm-blooded animal, such as ahuman or a dog. Such diseases include infections (which may be, forexample, bacterial, viral or protozoan) or diseases such as cancer. Inone embodiment, the disease is leishmaniasis, and the patient maydisplay clinical symptoms or may be asymptomatic. In general, theresponsiveness of a particular disease to IL-12 stimulation may bedetermined by evaluating the effect of treatment with a pharmaceuticalcomposition or vaccine of the present invention on clinical correlatesof immunity. For example, if treatment results in a heightened Th1response or the conversion of a Th2 to a Th1 profile, with accompanyingclinical improvement in the treated patient, the disease is responsiveto IL-12 stimulation. Polypeptide administration may be as describedbelow, or may extend for a longer period of time, depending on theindication. Preferably, the polypeptides employed in the pharmaceuticalcompositions and vaccines are complementary, as described above. Aparticularly preferred combination contains polypeptides that compriseimmunogenic portions of M15, Ldp23, Lbhsp83, Lt-1and LbeIF4A, Lmspla,Lmsp9a, and MAPS-1A. Soluble Leishmania antigens, with or withoutadditional polypeptides, may also be employed.

[0113] Routes and frequency of administration, as well as dosage, forthe above aspects of the present invention will vary from individual toindividual and may parallel those currently being used in immunizationagainst other infections, including protozoan, viral and bacterialinfections. In general, the pharmaceutical compositions and vaccines maybe administered by injection (e.g., intracutaneous, intramuscular,intravenous or subcutaneous), intranasally (e.g., by aspiration) ororally. Between 1 and 12 doses may be administered over a 1 year period.For therapeutic vaccination (i.e., treatment of an infected individual),12 doses are preferably administered, at one month intervals. Forprophylactic use, 3 doses are preferably administered, at 3 monthintervals. In either case, booster vaccinations may be givenperiodically thereafter. Alternate protocols may be appropriate forindividual patients. A suitable dose is an amount of polypeptide or DNAthat, when administered as described above, is capable of raising animmune response in an immunized patient sufficient to protect thepatient from leishmaniasis for at least 1-2 years. In general, theamount of polypeptide present in a dose (or produced in situ by the DNAin a dose) ranges from about 100 ng to about 1 mg per kg of host,typically from about 10 μg to about 100 μg. Suitable dose sizes willvary with the size of the patient, but will typically range from about0.1 mL to about 5 mL.

[0114] In another aspect, this invention provides methods for using oneor more of the polypeptides described above to diagnose Leishmaniainfection in a patient using a skin test. As used herein, a “skin test”is any assay performed directly on a patient in which a delayed-typehypersensitivity (DTH) reaction (such as induration and accompanyingredness) is measured following intradermal injection of one or morepolypeptides as described above. Such injection may be achieved usingany suitable device sufficient to contact the polypeptide orpolypeptides with dermal cells of the patient, such as a tuberculinsyringe or 1 mL syringe. Preferably, the reaction is measured at least48 hours after injection, more preferably 72 hours after injection.

[0115] The DTH reaction is a cell-mediated immune response, which isgreater in patients that have been exposed previously to a test antigen(i.e., an immunogenic portion of a polypeptide employed, or a variantthereof). The response may measured visually, using a ruler. In general,induration that is greater than about 0.5 cm in diameter, preferablygreater than about 1.0 cm in diameter, is a positive response,indicative of Leishmania infection, which may or may not be manifestedas an active disease.

[0116] The polypeptides of this invention are preferably formulated, foruse in a skin test, as pharmaceutical compositions containing at leastone polypeptide and a physiologically acceptable carrier, as describedabove. Such compositions typically contain one or more of the abovepolypeptides in an amount ranging from about 1 μg to 100 μg, preferablyfrom about 10 μg to 50 μg in a volume of 0.1 mL. Preferably, the carrieremployed in such pharmaceutical compositions is a saline solution withappropriate preservatives, such as phenol and/or Tween 80™.

[0117] The inventive polypeptides may also be employed in combinationwith one or more known Leishmania antigens in the diagnosis ofleishmaniasis, using, for example, the skin test described above.Preferably, individual polypeptides are chosen in such a way as to becomplementary to each other. Examples of known Leishmania antigens whichmay be usefully employed in conjunction with the inventive polypeptidesinclude K39 (Burns et al., Proc. Natl. Acad. Sci. USA, 1993 90:775-779).

[0118] The following Examples are offered by way of illustration and notby way of limitation.

EXAMPLES Example 1 Preparation of M15

[0119] This Example illustrates the preparation of a Leishmania antigenM15, having the sequence provided in SEQ ID NO:2.

[0120] An L. major (Friedlan strain) amastigote cDNA expression libraryprepared in the λZAP II vector (Stratagene, La Jolla, Calif.) wasscreened according to manufacturer's instructions using sera obtainedfrom L. major infected BALB/c mice (8 weeks post inoculation).Approximately 40,000 plaques were screened and four clones expressingreactive antigens were purified to homogeneity by two subsequent roundsof low density screening. Bluescript phagemid inserts were excised frompositive clones for further analysis. An EcoRI/SstII restrictionfragment from the 5′ end of one partial cDNA insert isolated duringfirst round screening (pLmal-1) was subsequently used as a probe torescreen for clones containing full length cDNA inserts. The probe waslabeled to high specific activity (˜10⁹ cpm/μg) with [α-³²P]dCTP usingthe random primer method and was used to screen ˜10,000 plaques of theL. major expression library described above. Positive clones werecompared by restriction enzyme digestion and the clone with the largestinsert (pfl1-1) was chosen for subsequent analysis.

[0121] DNA sequence analyses were performed on an Applied Biosystemsautomated sequencer using Taq polymerase and dye coupled ddNTPterminators or dye-labeled sequencing primers. The complete sequence ofthe 2685 bp insert was determined using a combination of primer-directedsequencing and by sequencing a series of overlapping Exonuclease IIIdeletion subclones generated using the Erase-a-base system (Promega,Madison, Wis.). The sequence of this insert is provided in SEQ ID NO: 1,and the deduced amino acid sequence is provided in SEQ ID NO:2.

[0122] The complete insert of clone pf1-1 was excised by digestion withBamHI/KpnI and was subcloned in frame into BamHI/KpnI digested pQE31(QUIAGEN) to generate the construct pM151A. E. coli containing thisconstruct inducibly expressed high levels of the L. major antigenencoded by pfl1-1 (designated as M15) with the addition of a 6-histidinetag at the amino terminus. Large volume cultures (500 ml) of E. colihost cells containing the pM151 A construct were induced to expressrecombinant protein by the addition of 2 mM IPTG at mid-log phase ofgrowth. Growth was continued for 4 to 5 hours and bacteria were thenpelleted and washed once with cold PBS. Bacteria were resuspended in 20ml of lysis buffer (50 mM Na₂HPO₄, pH 8.0, 300 mM NaCl, 10 mMβ-mercaptoethanol) containing 20 mg of lysozyme and were lysed by a 1hour incubation at 4 ° C. followed by brief sonication. Insolublematerial was removed by centrifugation at 10,000× g for 10 minutes andalthough the recombinant protein was found to be evenly distributedbetween the soluble and insoluble fractions the insoluble material wasdiscarded at this point. Recombinant protein containing the aminoterminal histidine tag was affinity purified using Ni-NTA resin (QIAGEN)according to the manufacturer's recommendations. Briefly, 8 ml of Ni-NTAresin resuspended in lysis buffer was added to the soluble lysatefraction and binding was conducted with constant mixing for 1 hour at 4°C. The mixture was then loaded into a gravity flow column and thenon-binding material was allowed to flow through. The Ni-NTA matrix waswashed 3 times with 25 ml of wash buffer (50 mM Na₂HPO₄, pH 6.0, 300 mMNaCl, 10 mM β-mercaptoethanol) and bound material was eluted in 25 ml ofelution buffer (50 mM Na₂HPO₄, pH 5.0, 300 mM NaCl, 10 mMβ-mercaptoethanol). The eluted material was then dialyzed against 3changes of PBS, sterile filtered and stored at −20° C. The purifiedrecombinant protein was shown by SDS-PAGE analysis to be free of anysignificant amount of E. coli protein. A small number of bands of lowermolecular weight were assumed to be proteolytic products of the L. majorantigen based on their reactivity by western blot analysis. A high titrepolyclonal antisera against M15 was generated in rabbits by repeatedsubcutaneous injection of recombinant protein. Western blot analysis oflysates from L. major promastigotes and amastigotes using this antiseraindicated that the protein is constitutively expressed throughout theparasite lifecycle.

Example 2 Preparation of LDP23

[0123] This Example illustrates the preparation of a Leishmania antigenLdp23, having the sequence provided in SEQ ID NO:4.

[0124] A. Purification of MHC Class II-associated Peptides from P388D1Macrophages Infected with L. donovani

[0125] To ascertain that in vitro infection of macrophages would loadtheir MHC class II molecules with parasite peptides, initial experimentswere carried out to test the ability of L. donovani-infected macrophagecell line P388D1 to present parasite antigens to L. donovani specificT-cells. This macrophage cell line was chosen because it has the sameH-2 haplotype as the BALB/c mouse, which is a strain of mouse moderatelysusceptible to L. donovani infection and selected to conduct the in vivoexperiments. Using a proportion of 3-5 parasites per cell and an initialincubation at room temperature for 4-6 hours follows by 37° C. for 24-48hours, close to 90% of the macrophages were infected. The level of MHCclass II molecule expression, as determined by FACS analysis, indicatedthat infection did not cause an effect on the levels of MHC class IIexpression when compared to non-infected control cells.

[0126] To test the ability of the L. donovani-infected P388D1 cells topresent parasite antigens, macrophages were infected as indicated aboveand incubated at 26° C. for 6 hours, and then as 37° C. for either 24,48 or 72 hours. At each of these time points the non-adherent cells andfree parasites were washed out and the adherent cells were mechanicallydislodged, washed and fixed with paraformaldehyde. These cells were thenused as antigen presenting cells (APCs) for purified lymph node T-cellsfrom BALB/c mice immunized with L. donovani promastigotes. To generatethese anti-L. donovani specific T-cells, BALB/c mice (H-2d) of bothsexes (The Jackson Laboratory, Bar Harbor, Me.) were immunized at 8 to14 weeks of age in the rear foot pad with 5-10×10⁶ L. donovanipromastigotes emulsified in complete Freund's adjuvant (CFA) (DifcoLaboratories, Madison, Mich.) as described in Rodrigues et al., ParasiteImmunol. 14:49 (1992). The draining lymph nodes were excised 8 daysafter the immunization and T-cells were purified in an anti-mouse Igcolumn to remove the B cells, as described in Bunn-Moreno andCampos-Neto, J. Immunol. 127:427 (1981), followed by a passage through aSephadex G10 column to remove the macrophages.

[0127] Stimulation index was calculated by dividing the cpm obtained forthe cells cultured in the presence of infected P388D1 macrophages by thecpm obtained for the cells cultured in the presence of non-infectedmacrophages, but subjected to the same conditions as the infectedmacrophages. The results shown FIG. 1 indicate that L. donovani-infectedP388D1 macrophage process parasite antigens and that optimalpresentation occurs after 48 hours of infection. No stimulation of theT-cells by the non-infected macrophages was observed.

[0128] To isolate the MHC class II associated L. donovani peptides,P388D1 macrophages were infected with L. donovani promastigotes for aninitial incubation of 6 hours at room temperature. The cultures werethen transferred to 37° C. for the remainder of the 48 hour incubationperiod. At a ratio of 3-5 parasites per macrophage nearly 90% of themacrophages were infected after 24 hours of incubation at 37° C.

[0129] The MHC class II molecules were then affinity-purified.Approximately 1.5×10¹⁰ L. donovani-infected or an equal number ofnon-infected P388D1 macrophages were used for each purification. Thecells were harvested, washed with PBS and incubated for 30 minutes incold lysis buffer (PBS, 1% Nonidet P40, 25 mM iodoacetamide, 0.04%sodium azide, 1 mM aprotinin and 1 mM PMSF). The insoluble material wasremoved by centrifugation at 40,000 g for 1 hour and the supernatant wasrecycled overnight at 4° C. over a 5 ml anti-MHC class II molecules(H-2d) Sepharose column (Protein G Sepharose column to which themonoclonal antibody MK-D6 has been bound). Culture supernatants of MK-D6hybridoma cells (American Type Culture Collection, Rockville, Md.) wereemployed as the source for anti-MHC class II (H-2 d) monoclonalantibody. The column was washed with 50 ml of lysis buffer and then with50 ml of PBS containing 0.5% octyl glucopyranoside detergent. Boundmolecules were eluted from the column with 1M acetic acid in 0.2% NaCl.The MHC/peptide molecules were separated from the IgG (MK-D6 monoclonalantibody) using a Centricon 100 filter unit (Amicon Division, W.R. Grace& Co., Beverly, Mass.). The peptides were then dissociated from theclass II molecules by the addition of acetic acid to 2.5M, followed byseparation using a Centricon 10 filter unit. The resulting peptidepreparation, present in the low molecular weight sample, was then driedusing a speed vac concentrator (Savant Instrument Inc., Farmingdale,N.Y.).

[0130] The peptides were redissolved in 200 μl of 0.05% TFA andseparated by reverse-phase high performance liquid chromatography(RP-HPLC) using a 2.1 mm×25 cm Vydac C-18 column at a flow rate of 0.15ml/min employing a 1 to 30% acetonitrile gradient (60 min) followed by a30 to 60% gradient (30 min) and then a 60 to 80% gradient (90-110 min).Non-infected P388D1 cells were similarly processed to serve asbackground control for endogenous MHC class II associated peptides. FIG.2 shows a representative experiment; four distinct peaks which arepresent only in the material isolated from infected macrophages (panelB), and not in the material isolated from uninfected macrophages (panelA) are indicated.

[0131] Out of three independent peptide extractions, twenty fivedistinct HPLC peptide peaks were isolated from L. donovani-infectedmacrophages and were subjected to protein sequence analysis usingautomated Edman degradation on an Applied Biosystems 477 gas-phaseprotein sequencer. Protein sequence and amino acid analysis wereperformed by the W.M. Keck Foundation, Biotechnology ResourceLaboratory, Yale University, New Haven, Conn. In practically alldeterminations, no assignment could be made for the first position.Also, in most cases the definition of the amino acid residues of the10-15 positions was based on the quantitative dominance of one residueover others. Using this approach, the sequences obtained for severalpeptides showed the presence of 3-6 different residues in many of the10-15 sequence cycles analyzed for each determination, reflecting amixture of peptides. In addition, sequences could not be obtained forsome peaks because the peptides were blocked. Notwithstanding, threepeptides sequences were determined. Amino-acid sequences were searchedfor identity with proteins in the GenBank database using the GENPETP,PIR and SWISSPROT programs. The sequence data base analysis revealedthat one of the peptides was highly homologous toglyceraldehyde-3-phosphate dehydrogenase of various species. Anotherpeptide had homology with elongation factor of several species,including Leishmania. The third sequence was not clearly related to anyknown proteins, and is shown below:

[0132] XQXPQ(L/K)VFDEXX (SEQ ID NO:11).

[0133] B. Cloning and Sequencing of the Ldp23 Gene

[0134] In order to retrieve the L. donovani protein that was processedinto a peptide associated with the MHC class II molecules of infectedmacrophages, the peptide sequence of uncertain origin was chosen toguide the strategy for cloning the corresponding parasite gene. A DNAfragment was initially amplified from L. donovani promastigote cDNA byPCR. The sense primer was a peptide derived oligonucleotide (5′>GGAATTCCCCInCAGCTInGTInTTCGAC <3′) (SEQ ID NO:12) containing an EcoRIrestriction endonuclease site (underlined). The bases were selectedfollowing the preferential codon usage of L. donovani, as described inLangford et al., Exp. Parasitol. 74:360 (1992). Inosine was used for theresidues of positions 4, 6 and 7 because of the low codon usageassurance for the corresponding amino acids. In addition, thecarboxyl-terminal L-glutamic acid was not included for the design of theprimer. The antisense primer was a poly-thymidine oligonucleotide (oligodT, downstream primer) containing a XhoI restriction endonuclease site.

[0135] The gene fragment was amplified from a L. donovani promastigotecDNA preparation using the following reaction conditions: one cycle of 3min at 94° C. immediately followed by 35 cycles of 1 min at 94° C., 1min at 45° C. and 1 min at 72° C. The L. donovani cDNA was prepared from5×10⁷ washed promastigote forms harvested at the log growth phase (3days culture). The cDNA was obtained using an Invitrogen cDNA cycle™ kit(Invitrogen Co., San Diego, Calif.). Oligonucleotide primers weresynthesized by the DNA Synthesis Laboratory, Department of Pathology,Yale University School of Medicine.

[0136] The PCR products were analyzed by gel electrophoresis. Only oneband of approximately 300 bp was obtained. This fragment was cloned andits sequence confirmed the sequence of the peptide-based primerincluding the glutamic acid codon, deliberately not included in theprimer sequence.

[0137] The PCR amplified gene fragment was ligated into the pCR™ vectorusing the TA cloning system (Invitrogen Co., San Diego, Calif.).Transformants were selected in LB medium containing 100μg/ml ampicillinand the plasmid DNA was isolated using the Wizard™ Minipreps DNApurification kit (Promega Co., Madison, Wis.). Insert DNA was releasedwith the restriction enzymes EcoRI and XhoI (New England Biolabs,Beverly, Mass.), purified from an agarose gel electrophoresis andlabeled with ³²P using a random priming method (Megaprime Labeling Kit,Amersham Life Science, Buckinghamshire, England).

[0138] This DNA fragment was used as probe to screen a L. donovanipromastigote cDNA library as described in Skeiky et al., Infect. Immun.62:1643 (1994). An approximately 650 bp cDNA (Ldp23) was excised fromthe phagemid by in vivo excision using the Stratagene protocol. DNAsequencing was performed using the Sequenase version 2 system (DNAsequencing kit) in the presence or absence of 7-deaza-GTP (United StatesBiochemical, Cleveland, Ohio). The sequence is provided as SEQ ID NO:3,and shows complete homology with the original 300 bp PCR fragment. A 525bp open reading frame containing an ATG codon that follows the last 4bases of the spliced leader sequence and 3 stop codons adjacent to thepoly A tail was identified. This frame also codes the carboxyl terminalsequence (KVFDE) (SEQ ID NO:13) of the purified MHC class II associatedpeptide. The sequence analysis of the deduced protein sequence revealedone potential glycosylation site (Asn-Cys-Ser) at positions 68-70.

[0139] Sequence analysis was performed using the University of WisconsinGenetics Computer Group Programs and the GenBank and EMBL data bases ofprotein and DNA sequences. The search for homology of the Ldp23 genewith known sequences revealed no significant homology.

[0140] C. Bacterial Expression and Purification of Recombinant Protein

[0141] The recombinant L. donovani peptide donor protein was produced inE. coli transformed with the pGEX 2T expression vector in which theLdp23 gene was subcloned in frame. PCR was used to subclone the clonedgene in frame into the expression vector pGEX 2T. Primers containing theappropriate restriction site enzymes, initiation and termination codonswere: 5′>GGATCCATGGTCAAGTCCCACTACATCTGC <3′ (SEQ ID NO:14) for theupstream primer and 5′ >GAATTCAGACCGGATAGAAATAAGCCAATGAAA <3′ (SEQ IDNO:15) for the downstream primer (restriction sites of BamHI and EcoRIare underlined respectively). PCR conditions were as indicated above forthe amplification of the original peptide related DNA fragment. Thetemplate used was pBluescript plasmid containing the cloned gene fromthe cDNA library.

[0142] Overexpression of the recombinant fusion protein was accomplishedby growing the transformed E. coli (DH5α) and inducing the tac promoterwith 1 mM isopropyl-β-thiogalactopyranoside (IPTG) (Stratagene, LaJolla, Calif.). Cells were collected, centrifuged, and analyzed for thepresence of the fusion protein by SDS-PAGE. A glutathione-S-transferasefusion protein of 43-44 kD was produced, indicating a leishmanialprotein of approximately 18 kD, as glutathione-S-transferase (GST) has aMW of 26 kD. However, the fusion protein was very insoluble andtherefore could not be purified by affinity chromatography using aglutathione column. The use of low concentrations of detergents likeSDS, sarcosyl, deoxycolate, and octyl-glucopyranoside during theextraction steps was efficient to solubilize the protein butunfortunately prevented its binding to the glutathione column. Othermaneuvers, such as the growth of the E. coli and incubation andinduction of the tac promoter with IPTG at 33° C., did not improve theprotein solubility. However, the purification was achieved bypreparative SDS-PAGE. The band was visualized with 0.1M KCl, cut andelectroeluted from the gel followed by extensive dialysis against PBSand concentration on Centricon 10 filters.

[0143] Approximately 500 μg of purified protein was obtained. Thepurified protein is shown in FIG. 3. In panel A, E. coli (DH5α)transformed with the expression vector pGEX 2T containing the Ldp23 genewas grown in LB medium and the tac promoter was induced with IPTG for 3hours. The cells were pelleted, resuspended in loading buffer andsubmitted to SDS-PAGE (10%) under reducing condition. The gel wasstained with Coomassie blue. Lane 1 shows the uninduced E. coli and land2 shows the induced E. coli. The arrow indicates the recombinantprotein. Panel B shows the protein prepared as in panel A and submittedto a preparative SDS-PAGE. The band corresponding to the overexpressedrecombinant fusion protein was identified by KCl, cut out, electroelutedfrom the gel strip, dialyzed against PBS and submitted to analyticalSDS-PAGE (12%). Numbers on the left side indicate the molecular weightsof the markers. Attempts to further purify the leishmanial protein bycleaving it out from the fusion protein GST with thrombin wereunsuccessful.

[0144] D. Expression of Ldp23

[0145] To ascertain that the Ldp23 peptide is expressed in Leishmaniaorganisms, a Northern blot analysis was performed using RNA preparedfrom different promastigote growth phases (logarithmic and stationary)and from the amastigote form of these parasites.

[0146] The RNA was prepared from 2×10⁷ parasite cells using the MicroRNA isolation kit (Stratagene, La Jolla, Calif.) according to thecompany's recommended instructions. RNA was prepared from L. donovanipromastigotes (logarithmic growth phase); from L. major promastigotes(logarithmic and stationary growth phases); from L. amazonensis, bothpromastigotes (logarithmic and stationary growth phases) and amastigotespurified from CBA/J infected mice; and from L. pifanoi, bothpromastigotes (logarithmic and stationary growth phases) and amastigotes(from axenic culture medium). L. donovani (1S strain), L. amazonensis(MHOM/BR/77/LTB0016), L. major (MHOM/IR/79/LRC-L251) and L. pifanoi(MHOM/VE/60/Ltrod) promastigotes were grown and maintained at 26° C. inSchneider's medium containing 20% FCS and 50 μg/ml gentamicin. Theamastigote forms of L. amazonensis were obtained by differentialcentrifugation of a “pus-like” foot pad lesion of a CBA/J mouse infectedfor 6 months with this parasite. L. pifanoi amastigotes were obtainedfrom axenic culture as previously reported by Pan et al., J. Euk.Microbiol. 40:213 (1993).

[0147] The hybridization was carried out at 45° C. in the presence of50% formamide, 5× Denhardt's solution, 0.1% SDS, 100 μg/ml singlestranded salmon sperm DNA and 5× SSPE using 0.45 μm Nytran membranefilters (Schleicher & Schuell, Keene, NH). The probe was the ³²P labeledLdp23 gene.

[0148]FIG. 4 shows that one single RNA band of 680 bp was observed forall growth phases and forms of all tested Leishmania. Within FIG. 4, thenumbers 1, 2 and 3 refer to RNA obtained from promastigotes at thelogarithmic growth phase, promastigotes at the stationary growth phaseand amastigote forms, respectively, and the numbers on the left sideindicate the molecular weights of the markers in base pairs. This resultis consistent with the corresponding gene size (525 bp) and with themolecular weight of the expressed protein and points to the ubiquitousdistribution and expression of this gene within the genus Leishmania.

[0149] E. Induction of Anti-L. donovani Antibody Response in Mice andRabbits by Purified Recombinant Protein

[0150] In order to evaluate the immunogenicity of the recombinantleishmanial protein, and to investigate its expression in the parasites,mice and rabbits were immunized with the GST-fusion protein in CFA.BALB/c mice were immunized in the rear foot pad with 5-10 μg of proteinemulsified in CFA. Protein concentration was determined using theBio-Rad Protein Assay reagent (Bio-Rad Laboratories, Richmond, Calif.).The mice were boosted 7 days later with 5-10 μg of protein emulsified inincomplete Freund's adjuvant (IFA) inoculated into the peritonealcavity. The mice were bled 7 days after the second immunization. NewZealand white rabbits (Millbrook Farm, Amherst, Mass.) were immunizedaccording to the following protocol: one intramuscular (IM) injection of25-30 μg of purified recombinant protein emulsified in CFA into eachthigh on day one; one 1M injection of 25-30 μg of purified proteinemulsified in IFA into each shoulder on day 7; on day 15, 25-30 μg ofthe purified protein in PBS was injected into the subcutaneous tissue.The rabbit was bled 7 days after the last immunization.

[0151] Sera were prepared and the anti-Leishmania antibody response wasmeasured by Western blot analysis and by FACScan. In both cases L.donovani promastigotes were used as antigen. Approximately 2×10⁶ L.donovani promastigotes were grown in Schneider's medium for 3 days (logphase), were washed with PBS, lysed with SDS-PAGE loading buffer andsubmitted to electrophoresis under reducing conditions using a 15%polyacrylamide gel. The proteins were transferred onto 0.45μ Immobilon-Ptransfer membrane (Millipore Co., Bedford, Mass.) using a wet-typeelectroblotter (Mini Trans-Blot Electrophoretic Transfer Cell, Bio RadLife Science Division, Richmond, Calif.) for 2 hours at 50 V. Themembranes were blocked overnight at room temperature with PBS containing3% normal goat serum (NGS), 0.2% Tween-20 and 0.05% sodium azide,followed by 3 washes with PBS. The blots were then incubated for 3-4hours at 4° C. with a {fraction (1/200)} dilution of pre-immune rabbitserum (lane A, FIG. 5) or with the same dilution of anti-fusion proteinrabbit antiserum (lane B, FIG. 5). The sera was previously absorbed 2×with non-viable desiccated Mycobacterium tuberculosis H-37 RA (DifcoLaboratories, Detroit, Mich.) and were diluted in PBS containing 1% NGSand 5% powdered non-fat bovine milk (Carnation, Nestlé Food Company,Glendale, Calif.). The membranes were then washed with PBS, incubatedfor 1 hour at room temperature with goat anti-rabbit IgG antibodyconjugated with alkaline phosphatase (Promega, Madison, Wis.), washedonce with PBS and 2× with veronal buffer pH 9.4. The reaction wasvisualized using the substrate mixture5-bromo-4-chloro-3-indoyl-phosphate and nitroblue tetrazolium(Kirkegaard & Perry Laboratories Inc., Gaithersburg, Md.) according tothe manufacturer's instructions.

[0152]FIG. 5 shows that the rabbit anti-recombinant protein antiserumdetects a single protein of 23 kDa (Ldp23) in the Leishmania crudeextract antigen preparation. No bands were observed when an anti-GSTantiserum was used (not shown). Moreover, the FACScan analysis (FIG. 6)shows that the antibody induced by the recombinant Ldp23 reacts withintact live L. donovani promastigotes, thus pointing to a cell surfaceexpression of this molecule on these organisms. The dotted line in FIG.6 shows the indirect immunofluorescence performed using pre-immune mouseserum and the solid line in FIG. 6 shows the result obtained with mouseanti-GST-Ldp23 antiserum. Both sera were diluted at {fraction (1/100)}.Parasites were washed with staining buffer and incubated with FITCconjugated goat anti-mouse immunoglobulin antibody. Fluorescenceintensity was analyzed by FACScan.

[0153] F. Recognition of Recombinant Ldp23 by Leishmania-specific LymphNode T-cells

[0154] To test the responsiveness of T-cells to the Ldp23 protein, twosets of experiments were performed. In the first experiment, lymph nodeT-cells (10⁵/well) from BALB/c mice immunized with L. donovanipromastigotes (as described above) were stimulated to proliferate with2×10⁵ Mitomycin C-treated normal mononuclear spleen cells (APC) andpulsed with the purified recombinant fusion protein. Proliferation ofT-cells was measured at 72 hours of culture. Values are expressed inFIG. 7 as cpm and represent the mean of [³H]TdR incorporation oftriplicate cultures. Background cpm of cells (T cells+APC) cultured inthe presence of medium alone was 1291. FIG. 7 shows that Leishmaniaspecific T-cells proliferate well and in a dose response manner torecombinant Ldp23. No response was observed when purified GST was addedinstead of the recombinant fusion protein nor when lymph node T-cellsfrom mice immunized with CFA alone were stimulated to proliferate in thepresence of the Leishmanial fusion protein (not shown).

[0155] The recognition of the recombinant Ldp23 protein byLeishmania-specific T-cells was also tested using two murine models ofleishmaniasis, the L. major highly susceptible BALB/c mice and the L.amazonensis susceptible CBA/J mice as described in Champsi andMcMahon-Pratt, Infect. Immun. 56:3272 (1988). These models were selectedto investigate the cytokine pattern induced by Ldp23. In the mouse modelof leishmaniasis, resistance is associated with Th 1 cytokines whilesusceptibility is linked to Th 2 responses.

[0156] Lymph node cells were obtained 3 weeks after the initiation ofinfection of BALB/c mice with L. major and the ability of these cells torecognize the recombinant Ldp23 was measured by proliferation and by theproduction of the cytokines IFN-γ and IL-4. 2×10⁶ cells obtained fromthe draining popliteal lymph node of infected mice were cultured for 72hours in the presence of recombinant Ldp23 or Leishmania lysate. Thelevels of IFN-γ and IL-4 in culture supernatants were measured by ELISAas previously described (Chatelain et al., J. Immunol. 148:1172 (1992),Curry et al., J. Immunol. Meth. 104:137 (1987), and Mossman and Fong, J.Immunol. Meth. 116:151 (1989)) using specific anti IFN-γ and IL-4monoclonal antibodies (PharMingen, San Diego, Calif.).

[0157] Ldp23 did stimulate these cells to proliferate (not shown) andinduced a typical Th 1 type of cytokine response as indicated by theproduction of high levels of IFN-γ (panel A of FIG. 8) and no IL-4(panel B of FIG. 8). Stimulation of these cells with a Leishmania crudelysate yielded a mixed Th cytokine profile. Exactly the same pattern ofcytokine production was obtained from the CBA/J mice infected with L.amazonensis (not shown). These results clearly indicate that Ldp23 is apowerful and selective activator of the Th 1 cytokines by mouse cells.

Example 3 Preparation of HsP83

[0158] This Example illustrates the preparation of a Leishmania antigenHsp83, having the sequence provided in SEQ ID NO:6.

[0159] A genomic expression library was constructed with sheared DNAfrom L. braziliensis (MHOM/BR/75/M2903) in bacteriophage λZAP II(Stratagene, La Jolla, Calif.). The expression library was screened withEscherichia coli preadsorbed serum from an L. braziliensis-infectedindividual with ML. Immunoreactive plaques were purified, and thepBSK(−) phagemid was excised by protocols suggested by the manufacturer.Nested deletions were performed with exonuclease III to generateoverlapping deletions for single-stranded template preparations andsequencing. Single-stranded templates were isolated following infectionwith VCSM13 helper phage as recommended by the manufacturer (Stratagene,La Jolla, Calif.) and sequenced by the dideoxy chain terminator methodor by the Taq dye terminator system using the Applied Biosystemsautomated sequencer model 373A.

[0160] Recombinant antigens produced by these clones were purified from500 ml of isopropyl-β-D-thiogalactopyranoside (IPTG)-induced cultures asdescribed in Skeiky et al., J. Exp. Med. 176:201-211 (1992). Theseantigens were then assayed for the ability to stimulate PBMC fromLeishmania-infected individuals to proliferate and secrete cytokine.Peripheral blood was obtained from individuals living in an area (Cortede Pedra, Bahia, Brazil) where L. braziliensis is endemic and whereepidemiological, clinical, and immunological studies have been performedfor over a decade, and PBMC were isolated from whole blood by densitycentrifugation through Ficoll (Winthrop Laboratories, New York, N.Y.).For in vitro proliferation assays, 2×10⁵ to 4×10⁵ cells per well werecultured in complete medium (RPMI 1640 supplemented with gentamicin,2-mercaptoethanol, L-glutamine, and 10% screened pooled A+ human serum;Trimar, Hollywood, Calif.) in 96-well flat-bottom plates with or without10 μg of the indicated antigens per ml or 5 μg of phytohemagglutinin perml (Sigma Immunochemicals, St. Louis, Mo.) for 5 days. The cells werethen pulsed with 1 μCi of [³H]thymidine for the final 18 h of culture.For determination of cytokine production 0.5 to 1 ml of PBMC wascultured at 1×10⁶ to 2×10⁶ cells per ml with or without the Leishmaniaantigens for 48 and 72 h.

[0161] The supernatants and cells were harvested and analyzed forsecreted cytokine or cytokine mRNAs. Aliquots of the supernatants wereassayed for gamma interferon (IFN-γ), tumor necrosis factor alpha(TNF-α), interleukin-4 (IL-4), and IL-10 as described in Skeiky et al.,J. Exp. Med. 181:1527-1537 (1995). For cytokine mRNA PCR analysis, totalRNA was isolated from PBMC and cDNA was synthesized by using poly(dT)(Pharmacia, Piscataway, N.J.) and avian mycloblastosis virus reversetranscriptase. Following normalization to β-actin, diluted cDNA wasamplified by PCR using Taq polymerase (Perkin-Elmer Cetus, Foster City,Calif.) with 0.2 μM concentrations of the respective 5′ and 3′ externalprimers in a reaction volume of 50 μl. The nucleotide sequences of theprimary pairs and the PCR conditions used were as described in Skeiky etal., J. Exp. Med. 181:1527-1537 (1995). We verified that our PCRconditions were within the semiquantitative range by initiallyperforming serial dilutions of the cDNAs and varying the number ofcycles used for PCR. Plasmids containing the human sequences for IL-2,IFN-γ, IL-4, IL-10, and β-actin were digested, and the DNA inserts werepurified after separation on 1% agarose gels. Radiolabeled ³²P probeswere prepared by the random priming method. PCR products were analyzedby electrophoresis on 1.5% agarose gels, transferred to nylon membranes,and probed with the appropriate ³²P-labeled DNA insert.

[0162] A recombinant clone was identified in the above assays which,following sequence comparison of its predicted amino acid sequence withsequences of other proteins, was identified as a Leishmania braziliensishomolog of the eukaryotic 83 kD heat shock protein (Lbhsp83). Thesequence of the clone is provided in SEQ ID NO:5 and the deduced proteinsequence is provided in SEQ ID NO:6. On the basis of the homology, thisclone, designated Lbhsp83a, appears to lack the first 47 residues of thefull length 703 amino acid residues. Lbhsp83 has an overall homology of94% (91% identity and 3% conservative substitution), 91% (84% identityand 7% conservative substitution) and 77% (61% identity and 16%conservative substitution) with L. amazonensis hsp83, T. cruzi hsp83 andhuman hsp89, respectively. A second clone (designated Lbhsp83b), whichcontained the 43 kD C-terminal portion of hsp83 (residues 331 to 703)was also isolated. FIG. 19 presents a comparison of the Lbhsp83 sequencewith L. amazonensis hsp83(Lahsp83), T. cruzi hsp83 (Tchsp83) and humanhsp89 (Huhsp89).

[0163] The results of proliferation assays using Lbhsp83a are shown inTable 1. Cells from all mucosal leishmaniasis (ML) patients proliferatedstrongly in response to Lbhsp83a, with stimulation indices (SIs) rangingfrom 19 to 558 (as compared to 20 to 1,634 for parasite lysate).Proliferation of PBMC from cutaneous leishmaniasis (CL) patients wasvariable and except for levels in two patients (IV and VII), levels weresignificantly lower than those of ML patients. By comparison, theproliferative responses of individuals with self-healing CL to Lbhsp83awere similar to those of individuals with ML. However, the responses ofall six self-healing individuals to Lbhsp83 were consistently higherthan those to Lbhsp83b. This suggests that PBMC from self-healing CLpatients preferentially recognize one or more T-cell epitopes locatedwithin the amino portion of Lbhsp83. TABLE 1 In vitro Proliferation ofPMBC from L. braziliensis-infected Individuals in Response to Lbhsp83Mean [³H]thymidine Group and incorporation [10³ cpm (SD)], SI with:Patient Lysate Lbhsp83a Lbhsp83b ML I 41.3, (1.3), 294 32.5, (6.6), 22146.7, (1.4), 318 II 44.2, (0.5), 104 20, (3.7), 47 36.7, (0.76), 86 III27.4, (1.5), 150 8.1, (1.7), 44 9.9, (0.32), 54 IV 52.7, (3.3), 13854.1, (6.2), 142 32.0, (1.3), 84 V 140.6, (7.6), 308 151.8, (57), 333150.4, (7.9), 331 VI 15.8, (1.8), 20 21.3, (4.4), 28 14.4, (1.3), 19 VII300.1, (9.4), 1634 102.1, (7.6), 558 41.7, (4.9), 228 CL I 0.26, (0.0),1.5 0.57, (0.3), 3.3 0.43, (0.17), 3.3 II 55.63, (8.6), 218 0.42, (0.0),1.6 0.8, (0.14), 3.2 III 0.39, (0.5), 4.0 3.4, (0.5), 9 2.6, (0.9), 6.6IV 19.14, (1.3), 87 7.17, (0.6), 32 5.9, (0.9), 27 V 0.32, (0.2), 3.01.47, (0.5), 14 0.3, (0.1), 3.0 VI 0.77, (0.1), 4.7 1.44, (0.2), 9 1.3,(0.6), 8.0 VII 4.01, (1.0), 2.0 60.3, (8.5), 15 66.7, (3.9), 16.6Self-healing CL I 19.7, (4.4), 94 61.3, (4.6), 293 5.0, (2.0), 24 II0.6, (0.1), 6.5 7.0, (2.0), 79 1.2, (0.8), 13 III 59.6, (7.1), 519 49.4,(3.1), 429 21.4, (3.7), 186 IV 0.2, (0.1), 1.6 13.1, (1.7), 108 0.6,(0.1), 5 V 27.1, (2.0), 225 6.3, (2.6), 52 3.0, (1.5), 25 VI 130.3,(14), 340 28.2, (2.9), 74 7.7, (3.8), 20 Control (uninfected) I 0.19,(0.0), 1.4 0.18, (0.0), 1.3 0.40, (0.16), 2.8 II 0.31, (0.1), 1.7 0.19,(0.0), 1.0 0.27, (0.0), 1.5 III 0.44, (0.2), 4.1 0.48, (0.1), 5.0 0.51,(0.2), 5.2 IV 0.4, (0.1), 3.2 0.52, (0.2), 5.1 0.50, (0.1), 5.0

[0164] A more detailed analysis of cytokine patterns of PBMC from MLpatients was performed by reverse transcriptase PCR. Cytokine mRNAs wereevaluated in cells prior to culturing (FIG. 9, lanes O) or followingculturing in the absence (lanes −) or presence of the indicated antigenfor 48 and 72 h. FIG. 4A shows the results for five of the six MLpatients whose PBMC were analyzed. In about half of the ML patients,noncultured (resting) PBMC had detectable levels of mRNA for IFN-γ,IL-2, and IL-4 but not IL-10. CL patient PBMC, however, had IL-10 mRNAin the resting state in addition to mRNAs for the other cytokines tested(FIG. 4B). Following in vitro culture without antigen, the levels ofmRNA for IFN-γ, IL-2, and IL-4 in resting cells from ML patientsdecreased to background levels while IL-10 mRNA levels increased. Incontrast, PBMC of most CL patients had stable or increased IL-10 mRNA,while the mRNAs for IL-2, IFN-γ, and IL-4 were reduced to barelydetectable levels in the absence of antigen stimulation.

[0165] In PBMC of three ML patients, stimulation with lysate resulted inincreased expression of mRNA for IFN-Y, IL-2, and IL-4 but not IL-10. Bycomparison, both Lbhsp83 polypeptides elicited the production of mRNAfor IFN-γ and IL-2 from all ML patient PBMC tested. In contrast,profiles of mRNA for IL-10 and IL-4 differed for the two hsp83polypeptides. Lbhsp83a stimulated the production of IL-10 but not IL-4mRNA (patients I, II, III, and IV), while Lbhsp83b stimulated theproduction of IL-4 but not IL-10 mRNA in all six patients.

[0166] All CL patients tested responded to both Lbhsp83 polypeptides aswell as to the parasite lysate by upregulating the synthesis of mRNAsfor IL-2 and IFN-γ, and in two of four patients (I and IV), the level ofIL-4 mRNA also increased, indicating stimulation of both Th1 and Th2cytokines. Interestingly and as in the case of ML patient unculturedPBMC which did not have detectable levels of IL-10 mRNA, Lbhsp83a andnot Lbhsp83b stimulated PBMC from one CL patient (IV) to synthesizeIL-10 mRNA. However, in the other three patients (I, II, and III) withresting levels of IL-10 mRNA, both rLbhsp83 polypeptides as well as theparasite lysate downregulated the expression of IL-10 mRNA.

[0167] PBMC supernatants were also assayed for the presence of secretedIFN-γ, TNF-α, IL-4, and IL-10. Cells from all ML and self-healing CLpatients (seven and six patients, respectively) and from four of sevenCL patients were analyzed for secreted IFN-γ following stimulation withboth rLbhsp83 polypeptides, parasite lysate and Lbhsp70, an L.braziliensis protein homologous to the eukaryotic 70 kD heat shockprotein (FIG. 10A). In general, rLbhsp83a stimulated patient PBMC tosecrete higher levels of IFN-γ than did rLbhsp83b (0.2 to 36 and 0.13 to28 ng/ml, respectively). The presence of secreted IFN-γ correlated wellwith the corresponding mRNA detected by PCR.

[0168] PBMC from four of five ML patients (I, II, V, and VII) hadsupernatant TNF-α levels (0.8 to 2.2 ng/ml) higher than those detectedin cultures of PBMC from uninfected controls following stimulation withparasite lysate (FIG. 10B). Similarly, the same PBMC were stimulated byrLbhsp83 to produce levels of TNF-α in supernatant ranging from 0.61 to2.9 ng/ml. Compared with those of uninfected controls, PBMC from three(I, V, and VI), five (I, II, IV, V, and VI), and two (II and V) of sixindividuals analyzed produced higher levels of TNF-α in response toparasite lysate, rLbhsp83a, and rLbhsp83b, respectively. The levels ofTNF-α produced by PBMC from CL patients in response to parasite lysatewere comparable to those produced by uninfected controls. However,rLbhsp83 stimulated TNF-α production in the PBMC of two of thesepatients. rLbhsp83a stimulated higher levels of TNF-α, production thandid rLbhsp83b. In the absence of antigen stimulation, only PBMC from MLpatients (five of six) produced detectable levels of supernatant TNF-α(60 to 190 pg/ml).

[0169] In agreement with the IL-10 mRNA, IL-10 was detected by ELISA inthe antigen-stimulated PMBC culture supernatants from ML and CLpatients. The levels (49 to 190 pg) were significantly higher (up to10-fold) following stimulation with rLbhsp83a compared with those afterparallel stimulation of the same cells with rLbhsp83b (FIG. 11).Parasite lysate also stimulated PMBC from some of the patients toproduce IL-10. Although rLbhsp83 stimulated PMBC from uninfectedindividuals to produce IL-10, with one exception, the levels were lowerthan those observed with patient PMBC. IL-4 was not detected in any ofthe supernatants analyzed. Therefore, the level of any secreted IL-4 isbelow the detection limit of the ELISA employed (50 pg/ml). Takentogether, the results demonstrate that a predominant Th1 -type cytokineprofile is associated with PMBC from L. braziliensis-infectedindividuals following stimulation with rLbhsp83 polypeptides.

[0170] To determine the correlation between the observed T-cellresponses and antibody production to Lbhsp83, we compared the antibody(immunoglobulin G) reactivities to Lbhsp83 in sera from the threepatient groups (FIG. 12). The ELISA reactivities of ML patient sera withrLbhsp83a were comparable to those observed with parasite lysate, and ingeneral, there was a direct correlation between ML patient anti-Lbhsp83antibody titer and T-cell proliferation. Of 23 serum samples from MLpatients analyzed, 22 were positive (˜96%) with absorbance values of0.20 to >3.0. Eleven of the ML patient serum samples had optical densityvalues that were >1. In general, CL patients had significantly loweranti-Lbhsp83 antibody titers (

=0.74; standard error of the mean [SEM]=0.1) compared to those of MLpatients. Therefore, ML and CL patient anti-rhsp83 antibody titerscorrelated with their respective T-cell proliferative responses.Anti-rLbhsp83 antibody titers were significantly higher in patients withML (

=1.5; SEM=0.2) than in self-healing CL patients (

=0.35; SEM=0.056), although their T-cell proliferative responses weresimilar. In fact, anti-Lbhsp83 antibody titers in serum fromself-healing CL patients were comparable to those from uninfectedcontrols (

=0.24; SEM=0.028). By using 2 standard deviations greater than the meanabsorbance value of uninfected control (0.484) as a criterion forpositive reactivity to Lbhsp83, eight of nine of the self-healingpatient serum samples tested were negative.

Example 4 Preparation Of Clones Encoding LT-210

[0171] This Example illustrates the preparation of clones encodingportions of the Leishmania antigen Lt-210, and which has the sequenceprovided in SEQ ID NO:8.

[0172] An expression library was constructed from L. tropica(MHOM/SA/91/WR1063C) genomic DNA. The DNA was isolated by solubilizingL. tropica promastigotes in 10 mM Tris-HCl, pH 8.3, 50 mM EDTA, 1% SDSand treating with 100 μg/ml RNaseA and 100 μg/ml proteinase K. Thesample was then sequentially extracted with an equal volume of phenol,phenol: chloroform (1:1), and Chloroform. DNA was precipitated by adding0.1 volume of 3M sodium acetate (pH 5.2) and 2.5 volume 95% ethanol. Theprecipitate was resuspended in 10 μM Tris, 1 mM EDTA. DNA was sheared bypassage through a 30-gauge needle to a size range of 2-6 kilobase, andwas repaired by incubation with DNA poli in the presence of 100 μM eachDATP, dCTP, dGTP, and dTTP. EcoRI adapters were ligated to the DNAfragments. After removal of unligated adapters by passage over a G-25Sephadex™ column, the fragments were inserted in EcoRI cut Lambda ZapII(Stratagene, La Jolla, Calif.).

[0173] Approximately 43,000 pfu were plated and screened with seraisolated from viscerotropic leishmaniasis (VTL) patients. Sera from VTLpatients were received from Drs. M. Grogl and A. Magill. The VTL patientgroup included eight individuals from whom parasites were isolated andcultured, seven of which had confirmed infection with L. tropica. Fourother patients were culture negative, but were still considered to beinfected based on either PCR analysis or a positive monoclonal antibodysmear (Dr. Max Grogl, personal communication). Serum samples from the 11infected patients were pooled and anti-E. coli reactivity removed byaffinity chromatography (Sambrook et al., supra, p. 12.27-12.28). Lambdaphage expressing reactive proteins were detected after antibody bindingby protein A-horseradish peroxidase and ABTS substrate.

[0174] Three clones, Lt-1, Lt-2, and Lt-3, containing a portion of theLt-210 gene were identified and purified. The clones ranged in size from1.4 to 3.3 kb and encoded polypeptides of 75 kD, 70 kD, and 120 kD,respectively. These three clones contain partial sequences of the Lt-210gene. Lt-1 and Lt-2 are overlapping clones and were chosen for furtherstudy.

[0175] The DNA sequences of Lt-1 and Lt-2 were determined. ExonucleaseIII digestion was used to create overlapping deletions of the clones(Heinikoff, Gene 28:351-359, 1984). Single strand template was preparedand the sequence determined with Applied Biosystems Automated Sequencermodel 373A or by Sanger dideoxy sequencing. The sequence on both strandsof the coding portion of Lt-1 clone was determined. The partial sequenceof one strand of Lt-2 clone was determined.

[0176] SEQ ID NO:7 presents the DNA sequence of Lt-1, and SEQ ID NO:8provides the predicted amino acid sequence of the open reading frame.The DNA sequence of the coding portion of the Lt-1 clone includes arepeated nucleotide sequence at the 5′ portion of the clone containingeight copies of a 99 bp repeat, three copies of a 60 bp repeat unit,which is part of the larger 99 bp repeat, and 800 bp of non-repeatsequence. The deduced amino acid sequence of the 99 bp repeat containslimited degeneracies. The mass of the predicted recombinant protein is67,060 Daltons. A database search of PIR with the predicted amino acidsequence of the open reading frame yielded no significant homology topreviously submitted sequences. Predicted secondary structure of therepeat portion of the clone is entirely α-helical.

[0177] Sequence analysis of Lt-2 revealed that the 3′ portion of theclone consisted of a mixture of 60 and 99 bp repeats that wereidentical, excepting occasional degeneracies, to the 60 and 99 bprepeats observed in Lt-1. Collectively, the sequencing data suggest thatLt-1 and Lt-2 are different portions of the same gene, Lt-2 beingupstream of Lt-1, with possibly a small overlap.

[0178] Hybridization analysis confirmed that rLt-2 and rLt-1 containoverlapping sequences. Genomic DNAs of various Leishmania species wererestricted with a variety of enzymes, separated by agarose gelelectrophoresis, and blotted on Nytran membrane filter (Schleicher &Schuell, Keene, N.H.). Inserts from rLt-1 and rLt-2 were labeled with³²P CTP by reverse transcriptase from random oligonucleotide primers andused as probes after separation from unincorporated nucleotides on aSephadex G-50 column. Hybridizations using the rLt-1 or the rLt-2 probeare performed in 0.2M NaH₂PO₄/3.6 M NaCl at 65° C., whereashybridization using the rLt-lr probe is performed in 0.2 M NaH₂PO₄/3.6 MNaCl/0.2 M EDTA at 60° C. overnight. Filters are washed in 0.075 MNaCl/0.0075 M sodium citrate pH 7.0 (0.15 M NaCl/0.0150 M sodium citratefor the Lt-lr probe), plus 0.5% SDS at the same temperature ashybridization.

[0179] Genomic DNA from a number of Leishmania species including L.tropica were analyzed by Southern blots as described above using theLt-1, Lt-2, and Lt-1r inserts separately as probes. Collectively,various digests of L. tropica DNA indicate that this gene has a low copynumber. A similar, overlapping pattern was observed using either theLt-1 or Lt-2 insert as a probe, consistent with the premise that thesetwo clones contain sequences near or overlapping one another. Inaddition, sequences hybridizing with these clones are present in otherLeishmania species.

[0180]L. tropica isolates have limited heterogeneity. Southern analysesof digested genomic DNA from four L. tropica parasite strains isolatedfrom VTL patients and three L. tropica parasite strains isolated from CLcases (two human, one canine) were performed. The Lt-1r insert describedbelow was labeled and used as a probe. The seven different L. tropicaisolates yielded similar intensities and restriction patterns, with onlya single restriction fragment length polymorphism among the isolates.These data, along with Southern analyses with additional enzymes,indicate limited heterogeneity in this region among the L. tropicaisolates.

[0181] The recombinant proteins of Lt-1 and Lt-2 were expressed andpurified. The nested deletion set of Lt-1 formed for sequencing includeda clone referred to as Lt-1r, which contains one and one-third repeats.This polypeptide was also expressed and purified. In vivo excision ofthe pBluescript SK⁻ phagemid from Lambda Zap II was performed accordingto the manufacturer's protocol. Phagemid virus particles were used toinfect E. coli XL-1 Blue. Production of protein was induced by theaddition of IPTG. Protein was recovered by first lysing pellets ofinduced bacteria in buffer (LB, 50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 10mM EDTA) using a combination of lysozyme (750 μg/mL) and sonication.rLt-1, rLt-2, and rLt-1r, were recovered from the inclusion bodies aftersolubilization in 8M urea (rLt-1 and rLt-2) or 4M urea (rLt-1r).Proteins rLt-1 and rLt-2 were enriched and separated by precipitationwith 25%-40% ammonium sulfate and rLt-1r was enriched by precipitationwith 10%-25% ammonium sulfate. The proteins were further purified bypreparative gel electrophoresis in 10% SDS-PAGE. Recombinant proteinswere eluted from the gels and dialyzed in phosphate-buffered saline(PBS). Concentration was measured by the Pierce (Rockford, Ill.) BCAassay, and purity assessed by Coomassie blue staining after SDS-PAGE.

Example 5 Preparation of LBEIF4A

[0182] This example illustrates the molecular cloning of a DNA sequenceencoding the L. braziliensis ribosomal antigen LbeIF4A.

[0183] A genomic expression library was constructed with sheared DNAfrom L. braziliensis (MHOM/BR/75/M2903) in bacteriophage λZAPII(Stratagene, La Jolla, Calif.). The expression library was screened withE. coli-preadsorbed patient sera from an L. braziliensis-infectedindividual with mucosal leishmaniasis. Plaques containing immunoreactiverecombinant antigens were purified, and the pBSK(−) phagemid excisedusing the manufacturer's protocols. Nested deletions were performed withExonuclease III to generate overlapping deletions for single strandedtemplate preparations and sequencing. Single stranded templates wereisolated following infection with VCSM13 helper phage as recommended bythe manufacturer (Stratagene, La Jolla, Calif.) and sequenced by thedideoxy chain terminator method or by the Taq dye terminator systemusing the Applied Biosystems Automated Sequencer Model 373A.

[0184] The immunoreactive recombinant antigens were then analyzed inpatient T-cell assays for their ability to stimulate a proliferative andcytokine production, as described in Examples 7 and 8 below.

[0185] A recombinant clone was identified in the above assays which,following sequence comparison of its predicted amino acid sequence withsequences of other proteins, was identified as a Leishmania braziliensishomolog of the eukaryotic initiation factor 4A (eIF4A). The isolatedclone (pLeIF.1) lacked the first 48 amino acid residues (144nucleotides) of the full length protein sequence. The pLeIF. 1 insertwas subsequently used to isolate the full length genomic sequence.

[0186] SEQ ID NO:9 shows the entire nucleotide sequence of thefull-length LbeIF4A polypeptide. The open reading frame (nucleotides 115to 1323) encodes a 403 amino acid protein with a predicted molecularweight of 45.3 kD. A comparison of the predicted protein sequence ofLbeIF4A with the homologous proteins from tobacco (TeIF4A), mouse(MeIF4A), and yeast (YeIF4A) shows extensive sequence homology, with thefirst 20-30 amino acids being the most variable. The lengths (403, 413,407, and 395 amino acids), molecular weights (45.3, 46.8, 46.4, and 44.7kDa), and isoelectric points (5.9, 5.4, 5.5, and 4.9) of LbeIF4A,TeIF4A, MeIF4A and YeIF4A, respectively, are similar. LbeIF4A shows anoverall homology of 75.5% (57% identity, 18.5% conservativesubstitution) with TeIF4A, 68.6% (50% identity, 18.6% conservativesubstitution) with MeIF4A and 67.2% (47.6% identity, 19.6% conservativesubstitution) with YeIF4A.

Example 6 Preparation of Soluble Leishmania Antigens

[0187] This Example illustrates the preparation of soluble Leishmaniaantigens from an L. major culture supernatant. L. major promastigoteswere grown to late log phase in complex medium with serum until theyreached a density of 2-3×10⁷ viable organisms per mL of medium. Theorganisms were thoroughly washed to remove medium components andresuspended at 2-3×10⁷ viable organisms per mL of defined serum-freemedium consisting of equal parts RPMI 1640 and medium 199, both fromGibco BRL, Gaithersburg, Md. After 8-12 hours, the supernatant wasremoved, concentrated 10 fold and dialyzed against phosphate-bufferedsaline for 24 hours. Protein concentration was then determined and thepresence of at least eight different antigens confirmed by SDS- PAGE.This mixture is referred to herein as “soluble Leishmania antigens.”

Example 7 Comparison of Intepleukin-4 and Interferon-γ ProductionStimulated by Leishmani Antigens

[0188] This Example illustrates the immunogenic properties of theantigens prepared according to Examples 1, 2, 5 and 6, as determined bytheir ability to stimulate IL-4 and IFN-γ in lymph node cultures frominfected mice and in human PBMC preparations. Lymph node cultures foruse in these studies were prepared from L. major-infected BALB/c mice 10days after infection, as described in Example 2. PBMC were preparedusing peripheral blood obtained from individuals with cured L. donovaniinfections who were immunologically responsive to Leishmania. Diagnosisof the patients was made by clinical findings associated with at leastone of the following: isolation of parasite from lesions, a positiveskin test with Leishmania lysate or a positive serological test.Uninfected individuals were identified based on a lack of clinical signsor symptoms, a lack of history of exposure or travel to endemic areas,and the absence of a serological or cellular response to Leishmaniaantigens. Peripheral blood was collected and PBMC isolated by densitycentrifugation through Ficoll™ (Winthrop Laboratories, New York).

[0189] Culture supernatants were assayed for the levels of secreted IL-4and IFN-γ. IFN-γ was quantitated by a double sandwich ELISA using mouseanti-human IFN-γ mAb (Chemicon, Temucula, Calif.) and polyclonal rabbitanti-human IFN-γ serum. Human rIFN-γ(Genentech Inc., San Francisco,Calif.) was used to generate a standard curve. IL-4 was quantitated insupernatants by a double sandwich ELISA using a mouse anti-human IL-4mAb (M1) and a polyclonal rabbit anti-human IL-4 sera (P3). Human IL-4(Immunex Corp., Seattle, Wash.) was used to generate a standard curveranging from 50 pg/ml to 1 ng/ml.

[0190]FIGS. 13A and 13B, illustrate the mean level of secreted IL-4 andIFN-γ, respectively, 72 hours after addition of 10 μg/mL of each of thefollowing antigens to a lymph node culture prepared as described above:soluble Leishmania antigen (i.e., an extract prepared from rupturedpromastigotes which contains membrane and internal antigens (SLA)),Ldp23, LbeIF4A (LeIF), Lbhsp83, M15 and LmeIF (the L. major homolog ofLbeIF4A). The levels of secreted IL-4 and IFN-γ in medium alone (i.e.,unstimulated) are also shown. While SLA elicits a predominantly Th2response from lymph node cells of Leishmania-infected mice, Ldp23,LbeIF4A, Lbhsp83 and M15 elicited relatively little IL-4 and largeamounts of IFN-γ, consistent with a Th1 response profile.

[0191]FIG. 14 shows the level of secreted IFN-γ in culture filtrate frominfected and uninfected human PBMC preparations 72 hours after additionof 10 μg/mL L. major lysate, M15 or L-Rack, an immunodominantleishmanial antigen in murine leishmaniasis. Similarly, FIG. 15illustrates the level of secreted IFN-γ in culture filtrate frominfected and uninfected human PBMC preparations 72 hours after additionof 10 μg/mL L. major lysate, soluble Leishmania antigens (prepared asdescribed in Example 6) or L-Rack. These results indicate that M15 andsoluble Leishmania antigens, but not L-Rack, are potent stimulators ofIFN-γ production in patient PBMC, but not in PBMC obtained fromuninfected individuals. Thus, M15 and soluble Leishmania antigens elicita dominant Th1 cytokine profile in both mice and humans infected withLeishmania.

Example 8 Comparison of Proliferation Stimulated by Leishmania Antigens

[0192] This Example illustrates the immunogenic properties of theantigens prepared according to Examples 1, 2, 5 and 6, as determined bytheir ability to stimulate proliferation in lymph node cultures frominfected mice and in human PBMC preparations.

[0193] For in vitro proliferation assays, 2-4×10⁵ cells/well werecultured in complete medium (RPMI 1640 supplemented with gentamycin,2-ME, L-glutamine, and 10% screened pooled A+ human serum; Trimar,Hollywood, Calif.) in 96-well flat bottom plates with or without 10μg/ml of the indicated antigens or 5 μg/ml PHA (Sigma Immunochemicals,St. Louis, Mo.) for five days. The cells were then pulsed with 1 μCi of[³H] thymidine for the final 18 hours of culture.

[0194]FIG. 16 illustrates the proliferation observed after addition of10 μg/mL or 20 μg/mL of each of the following antigens to a lymph nodeculture prepared as described in Example 7: SLA, Ldp23, LbeIF4A,Lbhsp83, and M15. The level of proliferation without the addition ofantigen is also shown. Data are represented as mean cpm. These resultsdemonstrate that a variety of leishmanial antigens are capable ofstimulatory lymph node cell proliferation from Leishmania-infected mice.

[0195]FIGS. 17 and 18 illustrate the proliferation observed in humanPBMC preparations from Leishmania-immune and uninfected individualsfollowing the addition of 10 μg/mL M15 and soluble Leishmania antigens,respectively. These values are compared to the proliferation observedfollowing the addition of culture medium, L. major lysate or L-Rack. Theresults show that M15 and soluble Leishmania antigens stimulateproliferation in Leishmania-immune PBMC, but not in PBMC obtained fromuninfected individuals, demonstrating that M15 and soluble antigens (butnot L-Rack) are recognized by PBMC from individuals immune to Leishmaniadue to a previous infection.

Example 9 Preparation of Lmsp1A and Lmsp9A

[0196] This Example illustrates the preparation of two solubleLeishmania antigens, Lmsp1a and Lmsp9a.

[0197] A. Purification of Lmsp1a and Lmsp9a from a Mixture of Soluble L.major Antigens

[0198] A high titer rabbit sera was raised against L. major solubleantigens, prepared as described above in Example 6. Specifically, a NewZealand white rabbit was immunized subcutaneously at multiple sites with180 μg of L. major soluble antigens in a suspension containing 100 μgmuramyl dipeptide and 50% incomplete Freund's adjuvant. Six weeks laterthe rabbit was given a subcutaneous boost of 100 μg of the same solubleantigen preparation in incomplete Freund's adjuvant. This was followedby two intravenous boosts spaced two weeks apart, each with 100 μg ofthe soluble antigen preparation. Sera was collected from the rabbit 11days after the final boost.

[0199] Anti E. coli antibody reactivities were removed from the rabbitsera by pre-adsorbing on nitrocellulose filters containing lysed E.coli. Adsorbed sera were evaluated by Western blot analysis using 10 μgLeishmania promastigote lysate (lane 1) and 1 μg soluble L. majorantigen mixture (lane 2). As shown in FIG. 20, the rabbit sera was foundto be reactive with seven dominant antigens of the soluble L. majorantigen mixture with molecular weights ranging from 18 to >200 kDa. Afour times longer exposure of the same blot revealed three additionalimmunoreactive species with molecular weights less than 18 kDa. The samesera reacted with approximately 10 antigens of the promastigote lysate,but with a pattern significantly different from that observed with thesoluble L. major antigens (FIG. 20). This is suggestive of potentialpost-translational modification of the same antigen before(intracellular localization) and after secretion/shedding. Suchmodifications may include cleavage of a leader sequence and/or theaddition of carbohydrate molecules to the secreted/shed antigens.

[0200] The rabbit sera described above was subsequently used to screenan L. major cDNA expression library prepared from L. major promastigoteRNA using the unidirectional Lambda ZAP (uni-ZAP) kit (Stratagene)according to the manufacturer's protocol. A total of 70,000 pfu of theamplified cDNA library was screened with the rabbit sera at a 1:250dilution. Nineteen positive clones were confirmed in the tertiaryscreening. The phagemid were excised and DNA from each of the 19 cloneswas sequenced using a Perkin Elmer/Applied Biosystems Division automatedsequencer Model 373A. All 19 clones were found to represent two distinctsequences, referred to as Lmsp1a and Lmsp9a. The determined cDNAsequences for Lmsp1a and Lmsp9a are provided in SEQ ID NO: 19 and 21,respectively, with the corresponding amino acid sequences being providedin SEQ ID NO: 20 and 22, respectively.

[0201] B. Characterization of Lmsp1a and Lmsp9a

[0202]FIG. 21 shows the full-length cDNA (SEQ ID NO: 19) and predictedamino acid sequence (SEQ ID NO: 20) for the antigen Lmsp1a. TheEcoRI/XhoI insert is 1019 bp long and contains the following features:a) the last 17 nt of the spliced leader sequence characteristic of alltrypanosoma nuclearly encoded mRNA; b) 39 nt of 5′ untranslatedsequence; c) an open reading frame of 453 nt long coding for a 151deduced amino acid sequence with a predicted molecular mass of 16.641kDa; and d) 471 nt of 3′ untranslated sequence terminating with a poly Atail. The predicted amino acid sequence contains three potentialphosphorylation sites at amino acid residues 3, 85 and 102. In addition,Lmsp1a contains an RGD sequence at residue 104, a sequence that may playa role in parasite invasion of the macrophage. RGD sequences have beenshown to mediate the binding of various adhesion proteins to their cellsurface receptors. There is no obvious leader sequence (secretorysignal) at the amino terminal portion suggesting that the protein mightbe shed or excreted. Lmsp1a appears to be one of the most abundantantigens found in the culture supernatant of live promastigote, since 17of the 19 clones contain sequences of variable lengths identical toLmsp1a.

[0203] Comparison of the amino acid sequence of Lmps1a with knownsequences using the DNA STAR system (Version 87) revealed that Lmsp1ashares between 65% to 70% homology with the eukaryotic nucleosidediphosphate kinase protein, also referred to in the mouse and human as atumor metastasis inhibitor gene.

[0204] Southern blot analysis of genomic DNA from L. major (Friedlanderstrain) digested with a panel of restriction enzymes (lanes 1 to 7) andsix other Leishmania species of different geographic locations digestedwith PstI (lanes 8 to 13) using the full-length cDNA insert of Lmps1a,demonstrated that Lmsp1a is present in all the species characterizedwith a high degree of conservation (FIG. 22). This suggests evolutionarysignificance for the maintenance of Lmsp1a and the existence ofhomologous species among all the Leishmania species.

[0205] The remaining two cDNA clones isolated from the soluble L. majorantigen mixture represent identical sequences (referred to as Lmsp9a;SEQ ID NO: 21), suggesting that the two copies resulted fromamplification of the primary library. Sequencing of the Lmsp9a cDNArevealed that the clone does not contain the full length 5′ sequencesince it is lacking both the spliced leader and 5′ untranslatedsequences. The 3′ end of the cDNA contains a poly A stretch, as would beexpected for a Leishmania mRNA. Of the predicted translated sequence(SEQ ID NO: 22), 34 of the 201 amino acids (17%) represent cysteineresidues. Comparison of the predicted protein sequence with those ofknown proteins as described above, revealed some homology with othercysteine rich proteins such as the major surface trophozoite antigen ofGiardia lamblia and furin proteases.

Example 10 Preparation and Characterization of MAPS-1A

[0206] This Example illustrates the preparation and characterization ofthe Leishmania antigen MAPS-1A (SEQ ID NO: 24).

[0207] A pool of sera was obtained from 5 BALB/c mice that had beengiven a primary immunization and two boosts with crude L. majorpromastigote culture supernatant as described below in Example 12. Thesemice were subsequently shown to be protected when challenged with a doseof live L. major promastigotes generally found to be lethal. The mousesera thus obtained were used to screen an L. major amastigote cDNAexpression library prepared as described in Example 1. Severalseroreactive clones were isolated and sequenced using a PerkinElmer/Applied Biosystems Division automated sequencer Model 373A (FosterCity, Calif.).

[0208] One of these clones, referred to herein as MAPS-1A, was found tobe full-length. Comparison of the cDNA and deduced amino acid sequencesfor MAPS-1A (SEQ ID Nos: 23 and 24, respectively) with known sequencesin the gene bank using the DNA STAR system revealed no significanthomologies to known Leishmania sequences, although some sequencesimilarity was found to a group of proteins, known as thiol-specificantioxidants, found in other organisms.

[0209] Recombinant MAPS-1A protein having an amino-terminal HIS-Tag wasprepared using a high level E. coli expression system and recombinantprotein was purified by affinity chromatography as described inExample 1. Southern blot analysis of genomic DNA from L. major digestedwith a panel of restriction enzymes, seven other Leishmania speciesdigested with PstI, and two other infectious-disease pathogens (T. cruziand T. brucei), using the full length insert of MAPS-1A, demonstratedthat MAPS-1A is present in all eight Leishmania species tested (FIG.23). Northern blot analysis of L. major promastigote and amastigote RNAsindicated that MAPS-1A is constitutively expressed.

[0210] Using oligonucleotide primers (SEQ ID NOs:27 and 28) based on theMAPS-1A cDNA sequence provided in SEQ ID NO: 23, the corresponding genewas isolated from L. tropica by means of PCR (using 30 cycles of thefollowing temperature step sequence: 94° C., 1 minute; 50° C., 1 minute;72° C., 1 minute) The determined cDNA sequence for the L. tropicaMAPS-1A protein is provided in SEQ ID NO: 25, with the correspondingamino acid sequence being provided in SEQ ID NO: 26.

[0211] The ability of recombinant MAPS-1A to stimulate cellproliferation was investigated as follows. PBMC from 3 L.braziliensis-infected patients having active mucosal leishmaniasis, from4 patients post kala-azar infection (previously infected with L. chagasiand/or L. donovani) and from 3 uninfected-individuals were prepared asdescribed above in Example 7. The ability of MAPS-1A to stimulateproliferation of these PBMC was determined as described in Example 8above. As shown in FIG. 24, significant levels of MAPS-1A specific PBMCproliferation were seen in 2 of the 7 Leishmania patients.

[0212] The ability of MAPS-1A to stimulate proliferation in mice lymphnode cultures was determined as described in Example 8. FIG. 25 showsthe amount of proliferation stimulated by MAPS-1A (at 25 μg/ml, 5 μg/mland 1 μg/ml) as compared to that stimulated by the positive control ConAand by crude L. major promastigote supernatant proteins, 20 dayspost-infection with L. major. Cells isolated 20 days post-infection werehighly responsive to MAPS-1A, whereas cells isolated 10 dayspost-infection were unresponsive.

Example 11 Immunoreactivity of Soluble Leishmania Antigens with Serafrom Leishmania-infected Patients

[0213] The reactivity of MAPS-1A with sera from uninfected individuals,from human leishmaniasis patients with cutaneous infection, from humanpatients with acute visceral leishmaniasis, and from L. major-infectedBALB/c mice was determined as follows.

[0214] Assays were performed in 96-well plates coated with 200 ngantigen diluted to 50 μL in carbonate coating buffer, pH 9.6. The wellswere coated overnight at 4° C. (or 2 hours at 37° C.). The platecontents were then removed and the wells were blocked for 2 hours with200 μL of PBS/1% BSA. After the blocking step, the wells were washedfive times with PBS/0.1% Tween 20™. 50 μL sera, diluted 1:100 inPBS/0.1% Tween 20™/0.1% BSA, was then added to each well and incubatedfor 30 minutes at room temperature. The plates were then washed againfive times with PBS/0.1% Tween 20™.

[0215] The enyzme conjugate (horseradish peroxidase—Protein A, Zymed,San Francisco, Calif.) was then diluted 1:10,000 in PBS/0.1% Tween20™/0.1% BSA, and 50 μL of the diluted conjugate was added to each welland incubated for 30 minutes at room temperature. Following incubation,the wells were washed five times with PBS/0.1% Tween 20™. 100 μL oftetramethylbenzidine peroxidase (TMB) substrate (Kirkegaard and PerryLaboratories, Gaithersburg, Md.) was added, undiluted, and incubated forabout 15 minutes. The reaction was stopped with the addition of 100 μLof 1 N H₂SO₄ to each well, and the plates were read at 450 mn.

[0216] As shown in FIG. 26, approximately 50% of the samples from humanleishmaniasis patients showed reactivities with recombinant MAPS-1Asubstantially above background. FIG. 27 shows the reactivity of MAPS-1Awith increasing dilutions of sera from BALB/c mice previouslyadministered either (i) saline solution; (ii) the adjuvant B. pertussis;(iii) soluble Leishmania antigens plus B. pertussis; (iv) live L. majorpromastigotes; or (v) soluble Leishmania antigens plus B. pertussisfollowed by live L. major promastigotes (as described below in Example12). Considerably higher absorbances were seen with sera from miceinfected with live L. major promastigotes and with mice infected withlive L. major promastigotes following immunization with solubleLeishmania antigens plus B. pertussis, than with sera from the otherthree groups of mice, indicating that anti-MAPS-1A antibody titersincrease following Leishmania infection.

Example 12 Use of Leishmania Antigens for Vaccination Against LeishmaniaInfection

[0217] This example illustrates the effectiveness of Leishmania antigensin conferring protection against disease in the experimental murineleishmaniasis model system. For a discussion of the murine leishmaniasismodel system see, for example, Reiner et al. Annu. Rev. Immunol.,13:151-77, 1995.

[0218] The effectiveness of (i) crude soluble Leishmania antigens, (ii)MAPS-1A, and (iii) a mixture of Ldp23, LbeIF4A and M15, as vaccinesagainst Leishmania infection was determined as follows. BALB/c mice (5per group) were immunized intra-peritoneally three times at biweeklyintervals with either (i) 30 μg crude soluble Leishmania antigens, (ii)20 μg MAPS-1A or (iii) a mixture containing 10 μg each of LeIF, Ldp23and M15, together with 100 μg of the adjuvant C. parvum. Two controlgroups were immunized with either saline or C. parvum alone. Two weeksafter the last immunization, the mice were challenged with 2×10⁵late-log phase promastigotes of L. major. Infection was monitored weeklyby measurement of footpad swelling. The amount of footpad swelling seenin mice immunized with either crude soluble Leishmania antigens, amixture of Ldp23, LbeiF4A and M15 (FIG. 28), or MAPS-1A (FIG. 29) wassignificantly less than that seen in mice immunized with C. parvumalone. These results demonstrate that the Leishmania antigens of thepresent invention are effective in conferring protection againstLeishmania infection.

Example 13 Isolation of DNA Encoding for Soluble Antigens from an L.major Genomic DNA Library

[0219] This example illustrates the isolation of seven solubleLeishmania antigen genes from an L. major genomic DNA library.

[0220] An L. major genomic DNA expression library was prepared from L.major promastigotes using the unidirectional Lambda ZAP (uni-ZAP) kit(Stratagene) according to the manufacturer's protocol. This library wasscreened with a high titer rabbit sera raised against L. major solubleantigens, as described above in Example 9. Seven positive clones wereidentified. The phagemid were excised and DNA from each of the sevenclones was sequenced using a Perkin Elmer/Applied Biosystems Divisionautomated sequencer Model 373A. The DNA sequences for these antigens,referred to as LmgSP1, LmgSP3, LmgSP5, LmgSP8, LmgSP9, LmgSP13, LmgSP19,are provided in SEQ ID NO:29-35, respectively, with the correspondingamino acid sequences being provided in SEQ ID NO: 36-42, respectively.LmgSP13 was found to contain a 39 amino acid repeat sequence shown inSEQ ID NO:43.

[0221] Subsequent studies led to the isolation of an extended cDNAsequence for LmgSP13 which contains an ORF (cDNA sequence provided inSEQ ID NO: 114) encoding a 194 amino acid sequence (SEQ ID NO: 119).Comparison of these sequences with those in the public databasesrevealed that LmgSP13 encodes a portion of a clone recently identifiedin the L. major genome sequencing project (genomic DNA sequence providedin SEQ ID NO: 115). The full-length ORF encodes a 2310 amino acidpolypeptide sequence (provided in SEQ ID NO: 120) containing uniqueamino- and carboxy-terminal regions flanking 42 highly related 39 aminoacid repeats.

[0222] The 5 repeats of the original LmgSP 13 clone were subcloned intoa modified pET vector both alone and as a fusion downstream of the M.tuberculosis antigen Ra12. The resulting recombinant protein wasexpressed, purified and used to generate a highly specific rabbitantiserum. Western blotting indicated that L. chagasi contains a LmgSp13homologue.

[0223] Subsequent studies resulted in the isolation of an extendedsequence for LmgSP9. The extended DNA sequence is provided in SEQ ID NO:54, with the corresponding predicted amino acid sequence being providedin SEQ ID NO: 55. The amino acid sequence was found to contain six 14amino acid repeat units (SEQ ID NO: 56), with each unit being furtherdivided into two 7 amino acid units, provided in SEQ ID NO: 57 and 58.Comparison of the isolated sequences for LmgSP9 with sequences in thepublic databases, revealed that LmgSP9 encodes for the carboy-terminalregion of a larger DNA sequence (SEQ ID NO: 116) that encodes a 708amino acid polypeptide (SEQ ID NO: 121) identified in the L. majorgenome sequencing project. LmgSP9 was found to share low homology withserine protease, endo-protease furin and the major surface-labeledtrophozoite antigen of Giardia lamblia (25-30% identity). Surfacelocalization of LmgSP9 is consistent with motif predictions of anamino-terminal signal sequence, carboxy-terminal transmembrane domainand GPI anchor. Southern hybridization using the original LmgSP9 cloneindicated that homologous sequences are present in all tested Leishmaniaspecies. The amino-terminal 295 amino acids of LmgSP9 excluding thesignal sequence (referred to as LmgSP9N-ht; cDNA sequence provided inSEQ ID NO: 117 and amino acid sequence provided in SEQ ID NO: 122) weresubcloned into a modified pET vector and recombinant protein wasexpressed and purified.

[0224] Comparison of the DNA and amino acid sequences for the isolatedantigens as described above, revealed no significant homologies toLmgSP1 and LmgSP3. LmgSP5 was found to be related to the knownPromastigote surface antigen-2 (PSA2) family. LmgSP8 was found to bearsome homology to a sequence previously identified in E. coli(2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylic acid synthase).LmgSP9 and LmgSP 19 were found to be homologous to a L. majorhydrophilic surface protein referred to as Gene B (Flinn, H. M. et al.Mol. Biochem. Parasit. 65:259-270, 1994), and to ubiquitin,respectively. To the best of the inventors' knowledge, none of theseantigens have been previously shown to elicit T or B cell responses.

[0225] In further studies, a 220 bp DNA fragment was amplified fromLmgSP5 and used to screen a L. major genomic library in Lambda ZAP.Seventeen positive clones were purified after secondary screening. Toselect for a clone that had a likelihood of having the 5′ sequence ofthe LmgSP5 insert, a labeled oligonucleotide from the 5′ region was usedto screen the DNA from the secondary positive clones. DNA from threeclones hybridized to the 5′ oligonucleotide, with one clone hybridizingstronger than the other two. This clone (cDNA sequence provided in SEQID NO: 103) was found to contain an insert of 2421 bp which containedthe entire open reading frame for the novel PSA-2 gene. This ORF wasamplified and cloned in the expression vector pET-17b for expression ofrecombinant protein in E. coli. The cDNA sequence of the ORF is providedin SEQ ID NO: 102, with the corresponding amino acid sequence beingprovided in SEQ ID NO: 104.

[0226] The reactivity of recombinant LmgSP9 with sera from patients withvisceral leishmaniasis, (from both Sudan and Brazil) and from normaldonors was evaluated by ELISA as described above. The absorbance valueswere compared with those obtained using the known Leishmania antigen K39described above, with L. chagasi lysate being employed as a positivecontrol. Representative results of these assays are provided below inTable 2, wherein all the patients from Brazil and those from the Sudandesignated as “VL” were inflicted with visceral leishmaniasis. Theresults demonstrated that LmgSP9 specifically detects antibody in mostindividuals with visceral leishmaniasis, regardless of geographicallocation. In several cases, the absorbance values of the antibodyreactivity to LmgSP9 were comparable to that observed with K39. Inaddition, LmgSP9 detected several cases of leishmaniasis that were notdetected using K39. These results indicate that LmgSP9 can be used tocomplement the reactivity of K39. TABLE 2 REACTIVITY OF LMGSP9 WITH SERAFROM LEISHMANIA PATIENTS Patient No. L. chagasi lysate K39 LmgSP9Sudanese samples: B19 1.067 0.306 0.554 B25 1.884 3.435 0.974 B43 1.19 3.225 0.86  B47 2.405 2.892 0.375 B50 0.834 0.748 0.432 B58 0.921 0.2350.92  B63 1.291 0.303 0.764 B70 0.317 0.089 3.056 VL4 1.384 3.035 2.965VL11 0.382 0.144 0.142 VL12 0.277 0.068 0.098 VL13 0.284 0.12  0.194Brazilian samples: 105 3.508 3.53  0.374 106 2.979 3.373 2.292 107 2.5353.444 0.46  109 1.661 3.415 3.319 111 3.595 3.537 0.781 112 2.052 3.4690.63  113 3.352 3.429 0.963 114 2.316 3.437 1.058 115 2.073 3.502 1.186116 3.331 3.461 0.96  Normal Donors: 129 0.157 0.104 0.08  130 0.1950.076 0.095 131 0.254 0.134 0.086 132 0.102 0.035 0.043

[0227] In subsequent ELISA analyses performed as described above, LmgSP13 was demonstrated to react as strongly with sera from patientsinfected with L. chagasi as with sera from L. major infected patients.This is consistent with the Western blot studies discussed above whereinL. chagasi was found to contain an LmgSp13 homologue.

[0228] In order to obtain a higher specificity for the detection ofantibodies in sera from visceral leishmaniasis patients, a homologue ofLmgSP9 was isolated from L. chagasi, one of the causative agents ofvisceral leishmaniasis. A total of 80,000 pfu of an amplified L. chagasigenomic library were screened with the entire coding region of LmgSP9(amplified from L. major genomic DNA). Seven hybridizing clones werepurified to homogeneity. The determined DNA sequences for two of theseclones, referred to as Lc Gene A and LcGene B, are provided in SEQ IDNO: 59 and 60, respectively, with the corresponding predicted amino acidsequences being provided in SEQ ID NO: 61 and 62, respectively. The openreading frame for Lc Gene A was found to show some homology to Gene A/C,previously isolated from L. major (McKlean et al., Mol. Bio. Parasitol.,85:221-231, 1997). The open reading frame for Lc Gene B showed somehomology to Gene B of L. major, discussed above, and was found tocontain eleven repeats of a 14 amino acid repeat unit (SEQ ID NO: 63),with each repeat being further divided into two 7 amino acid units,provided in SEQ ID NO: 64 and 65.

[0229] The diagnostic potentials of Lc Gene A and Lc Gene B wereevaluated by ELISA as described above using sera from visceralleishmaniasis patients from Sudan and Brazil, and from uninfectedcontrols. Absorbance values were compared to those obtained usingLmgSP9. Much higher absorbance values were obtained with Lc Gene A andLc Gene B than with LmgSP9, with Lc Gene B appearing to be moreeffective that Lc Gene A in detecting antibodies in certain cases. Theseresults indicate that Lc Gene B is highly effective in the diagnosis ofvisceral leishmaniasis.

[0230] In order to assess the diagnostic potential of the repeats foundwithin Lc Gene B, a series of 6 peptides were synthesized (SEQ ID NO:66-71; referred to as Pep 1-6), differing in an R or H residue. An ELISAwas carried out using the full-length LcGene B protein and the sixpeptides. The absorbance values obtained with Pep 3 were higher thanthose obtained with the other 5 peptides, however they were not as highas those obtained with the full length protein.

Example 14 Isolation and Characterization of DNA Encoding for SolubleAntigens from an L. chagasi Genomic DNA Library

[0231] This example illustrates the preparation of five solubleLeishmania antigen genes from an L. chagasi genomic DNA library.

[0232] An L. chagasi genomic DNA expression library was prepared from L.chagasi promastigotes using the unidirectional Lambda ZAP (uni-ZAP) kit(Stratagene) according to the manufacturer's protocol. This library wasscreened with a high titer rabbit sera raised against L. major solubleantigens, as described above in Example 9. Five positive clones wereidentified. The phagemid were excised and DNA from each of the Fiveclones was sequenced using a Perkin Elmer/Applied Biosystems Divisionautomated sequencer Model 373A. The DNA sequences for these antigens,referred to as LcgSP1, LcgSP3, LcgSP4, LcgSP8, and LcgSP10 are providedin SEQ ID NO:44-48, respectively, with the corresponding amino acidsequences being provided in SEQ ID NO:49-53, respectively.

[0233] Comparison of these sequences with known sequences in the genebank as described above, revealed no known homologies to LcgSP3, LcgSP4,LcgSP8 and LcgSP10. LcgSP1 was found to be homologous to the knownantigen HSP70.

[0234] Subsequent studies led to the isolation of a longer cDNA sequencefor LcgSP3. This clone was found to contain an ORF (cDNA sequenceprovided in SEQ ID NO: 113) encoding an amino acid sequence of 539residues (SEQ ID NO: 118). Comparison of the sequence for LcgSP3 withthose in the public database, revealed it to be most closely related tothe thermostable carboxypeptidase of Vibrio cholera (45% identity).Moreover, LcgSP3 was found to contain the active site residuescharacteristic of this class of carboxypeptidase. Southern hybridizationusing the LcgSP3 ORF indicated that homologous sequences are present inall tested Leishmania species. The LcgSP3 ORF was subcloned into amodified pET vector and recombinant protein was expressed, purified andused to generate a highly specific rabbit antiserum, using conventionaltechniques.

[0235]FIGS. 30A and B illustrate the proliferative response of murinelymph nodes to recombinant LcgSP8, LcgSP10 and LcgSP3. Lymph nodes weretaken BALB/c mice 17 days after infection with L. major. Infectionoccurred by footpad injection of 2×10⁶ parasites/footpad. The cells werestimulated with recombinant antigen and proliferation was measured at 72hours using ³H-thymidine. FIG. 30A shows the CPM, a direct measurementof mitotic activity in response to the antigens, and FIG. 30B shows thestimulation index, which measures the proliferative response relative tothe negative control.

Example 15 Isolation of DNA Encoding for L. major Antigens By CD4+ TCell Expression Cloning

[0236] This example illustrates the isolation of T cell antigens of L.major using a direct T cell screening approach.

[0237] Leishmania-specific CD4+ T cell lines were derived from the PBMCof an individual who tested positive in a leishmania skin test but hadno clinical history of disease. These T cell lines were used to screen aL. major amastigote cDNA expression library prepared as described inExample 1. Immunoreactive clones were isolated and sequenced asdescribed above. The determined cDNA sequences for the 8 isolated clonesreferred to as 1G6-34, 1E6-44, 4A5-63, 1B11-39, 2A10-37, 4G2-83, 4H6-41,8G3-100 are provided in SEQ ID NO: 72-79, respectively, with thecorresponding predicted amino acid sequences being provided in SEQ IDNO: 80-87, respectively. The cDNA sequences provided for 1E6-44,2A10-37, 4G2-83, 4H6-41 and 8G3-100 are believe to represent partialclones. All of these clones were shown to stimulate T cellproliferation.

[0238] Comparison of these sequences with those in the gene bank asdescribed above revealed no known homologies to the antigen 4A5-63.1G6-34 was found to have some homology to histone H2B previouslyidentified in L. enrietti. Antigens 1E6-44, 1B11-39 and 8G3-100 showedsome homology to sequences previously identified in other eukaryotes, inparticular Saccharomyces cerevisae. 2A10-37 and 4H6-41 were found to behomologous to the two previously identified proteins alpha tubulin fromL. donovani and beta tubulin from L. major, respectively, and 4G2-83 wasfound to be homologous to elongation initiation factor 2 previouslyidentified in T. cruzi.

[0239] Subsequent full-length cloning studies, using standardtechniques, led to the isolation of an extended cDNA sequence for1E6-44, provided in SEQ ID NO: 105. The corresponding amino acidsequence is provided in SEQ ID NO: 106. An extended cDNA sequence for2A10-37 is provided in SEQ ID NO: 107. This sequence was found tocontain a complete open reading frame which encodes the amino acidsequence of SEQ ID NO: 108. An extended cDNA sequence for 4G2-83 isprovided in SEQ ID NO: 109. This sequence contains a complete openreading frame which encodes the amino acid sequence of SEQ ID NO: 110.An extended cDNA sequence for 8G3-100 is provided in SEQ ID NO: 111,with the corresponding amino acid sequence being provided in SEQ ID NO:112.

[0240] All eight of the antigens described above (1G6-34, 1E6-44,4A5-63, 1B11-39, 2A10-37, 4G2-83, 4H6-41, 8G3-100) were expressed in E.coli as recombinant fusion proteins containing N-terminal histidine tagsand were purified to homogeneity using nickel affinity chromatography.All 8 purified recombinant proteins elicited strong proliferativeresponses from the CD4+ T cell lines employed in the library screening.T cell reactivity to 1G6-34, 4H6-41 and 8G3-100 was also observed in Tcells generated against both Leishmania promastigote culture filtrateand amastigote culture filtrate, indicating that these antigens areexpressed in both the promastigote and amastigote life stages at levelsthat are sufficient to evoke strong cellular immunes response.

[0241] The ability of the 8 antigens to stimulate proliferation andIFN-γ production in PBMC from patients with active cutaneousleishmaniasis (CL) and from normal donors was examined as describedabove. In addition to the 8 antigens, leishmanial promastigote lysate(LPr) and purified protein derivative from M. tuberculosis (PPD) werealso tested. The number of patients and/or donors responding to eachantigen is shown in Table 3 below. All CL patients responded to at leastone of the 8 antigens. Most notably, the antigens 1G6-34 and 4H6-41elicited cell proliferation in 6/7 and 7/7 CL patients, respectively,and IFN-γ production in 6/7 and 5/7 CL patients, respectively. Inaddition 1G6-34 was not recognized by PBMC from uninfected controldonors. TABLE 3 CELL PROLIFERATION AND IFN-γ PRODUCTION IN PBMC FROMPATIENTS WITH CUTANEOUS LEISHMANIASIS CL Patients Normal donors IFN-γCell IFN-γ Cell Antigen production Proliferation productionProliferation LPr 7/7 6/7 3/8 5/8 1G6-34 6/7 5/7 0/8 0/8 1E6-44 0/7 4/75/8 2/8 4A5-63 1/7 1/7 0/8 0/8 1B11-39 5/7 3/7 1/8 0/8 2A10-37 1/7 3/71/8 0/8 4H6-41 7/7 5/7 3/8 1/8 8G3-100 0/7 2/7 5/8 2/8 PPD 7/7 7/7 7/87/8

Example 16 Synthesis of Polypeptides

[0242] Polypeptides may be synthesized on a Perkin Elmer/AppliedBiosystems Division 430A peptide synthesizer using FMOC chemistry withHPTU (O-Benzotriazole-N,N,N,′,N′-tetramethyluronium hexafluorophosphate)activation. A Gly-Cys-Gly sequence may be attached to the amino terminusof the peptide to provide a method of conjugation, binding to animmobilized surface, or labeling of the peptide. Cleavage of thepeptides from the solid support may be carried out using the followingcleavage mixture: trifluoroaceticacid:ethanedithiol:thioanisole:water:phenol (40:1:2:2:3). After cleavingfor 2 hours, the peptides may be precipitated in coldmethyl-t-butyl-ether. The peptide pellets may then be dissolved in watercontaining 0.1% trifluoroacetic acid (TFA) and lyophilized prior topurification by C18 reverse phase HPLC. A gradient of 0%-60%acetonitrile (containing 0.1% TFA) in water (containing 0.1% TFA) may beused to elute the peptides. Following lyophilization of the purefractions, the peptides may be characterized using electrospray or othertypes of mass spectrometry and by amino acid analysis.

Example 17 Use of Leishmania Antigens Plus Adjuvant for VaccinationAgainst Leishmania Infection

[0243] This example illustrates the effectiveness of recombinantLeishmania antigens, M15 and MAPS, plus an adjuvant, IL-12, inconferring protection against disease in the experimental murineleishmaniasis model system. For discussion of the murine leishmaniasismodel system see, for example, Reiner et al., Annu. Rev. Immunol.,13:151 -77, 1995. The effectiveness of M15 and MAPS in combination withIL-12, as vaccine against Leishmania infection was determined asfollows: BALB/c mice (5 per group) were immunized subcutaneously in theleft footpad, twice (3 weeks apart) with the 10 μg of the individualantigens mixed with 1 μg of IL-12. As controls, three separate groups ofmice were immunized with soluble leishmania lysate antigens (SLA) plusIL-12, with IL-12 alone or with PBS. Three weeks after the lastimmunization the mice were infected in the right footpad with 2×10⁵promastigote forms of L. major (stationary phase). Footpad swelling wasthen measured weekly. Results are expressed in FIG. 31 and clearlyindicate that the mice immunized with either M15 or MAPS and IL-12 weregreatly protected against the infection; whereas mice immunized withIL-12 alone did not show protection from infection. The protectioninduced by these antigens was as efficient or better than that inducedby SLA+IL-12, a regimen known to induce good protection againstleishmaniasis in this animal model (Afonso, L. C. C., T. M. Scharton, L.Q. Vieira, M. Wysocka, G. Trinchieri, and P. Scott. 1994. The adjuvanteffect of interleukin-12 in a vaccine against Leishmania major. Science263:235-237). The same pattern of protection described above, wasobtained i.e., M15, MAPS, and SLA, induced protection against L. majorinfection when C. parvum instead of IL-12 was used as adjuvant (Example12). These results demonstrate that both M15 and MAPS recombinantantigens induce excellent protection against L. major infection in theBALB/c model of human leishmaniasis. In addition, both antigens inducedprotection when tested in two different adjuvant formulations, (e.g.,IL-12 and C. parvim.) This finding is of high significance because itdemonstrates that immunity to leishmaniasis can be induced by thespecific antigens delivered in adjuvants that are suitable for humanuse.

Example 18 Use of Leishmania DNA for Vaccination Against LeishmaniaInfection

[0244] This example illustrates the effectiveness of Leishmania DNA inconferring protection against disease in the experimental murineleishmaniasis model system. For discussion of the murine leishmaniasismodel system see, for example, Reiner et al., Annu. Rev. Immunol.,13:151-77, 1995. The protection properties of the recombinant antigenswas tested by immunizing mice with naked DNA containing thecorresponding M15 and MAPS genes. The DNA construct used was thepcDNA3.1 vector (Invitrogen) containing a CMV promotor. BALB/c mice (5per group) were injected in the left footpad three times (3 weeks apart)with 100 g of the indicated naked DNA preparations. Mice were bledbefore and after the immunizations to monitor the development ofspecific immune response. The antibody response was evaluated by ELISA.Specific anti-M15 and anti-MAPS IgG2a antibodies were detected after thesecond immunization in the sera of the mice immunized with therespective naked DNA. The presence of specific antibodies indicates thatthe DNA immunization resulted in the production of specific proteinantigen. Three weeks after the last immunization, the mice were thenchallenged in the right footpad with 2×10⁵ promastigote forms of L.major (stationary phase). Footpad swelling was then measured weeklythereafter. Results are expressed in FIG. 32 and clearly indicated that,again, mice immunized with naked DNA containing either the M15 or MAPSgenes were greatly protected against the infection with L. major. Theseresults demonstrate that both M15 and MAPS genes induce excellentprotection against L. major infection in the BALB/c model of humanleishmaniasis.

Example 19 Preparation and Characterization of Leishmania FusionProteins

[0245] Fusion proteins comprising the Leishmania antigens MAPS-iA (SEQID NO: 24), M15 (SEQ ID NO: 2), Lbhsp83 (SEQ ID NO: 6) and LbeIF4A (SEQID NO: 10) were prepared as follows.

[0246] A fusion construct of MAPS-1A and M15 was prepared by first PCRamplifying the full-length coding sequence of MAPS-1A using the primersof SEQ ID NO: 88 and 89. The resulting products were digested with NdeIand BamHI follows by sub-cloning into the pET17b expression vector, alsodigested with NdeI and BamHI. The ligated products were transformed intoE. coli and transformants containing the correct insert were identifiedby restriction digest and verified by DNA sequencing. The MAPS-1A-pETplasmid was digested with BamHI and EcoRI. The latter cuts within thepoly-linker sequence of the pET vector which is located downstream ofthe BamHI site.

[0247] The primers of SEQ ID NO: 90 and 91 were employed to PCR amplifythe full-length coding sequence of M15 and the resulting product wasdigested with BamHI and EcoRI followed by sub-cloning into thepredigested MAPS1A-pET plasmid above. The ligated products were thentransformed into E. coli and transformants with the correct insert wereidentified by restriction digest and verified by DNA sequencing. TheMAPS1A-M15 pET construct was transformed into the bacterial host (BL21;pLysE). Expression of the protein resulted in a single recombinantmolecule with a predicted molecular weight of 85.7 kDa. The recombinantMAPS1A-M15 fusion protein also contained 33 amino acid residues ofrun-through vector as a result of the removal of the stop codon of M15and was subsequently digested with EcoRI. The DNA sequence of theMAPS1A-M15 construct is provided in SEQ ID NO: 101.

[0248] The primers of SEQ ID NO: 92 and 93 were used to PCR amplify thefirst 226 amino acid residues of LbeIF4A. The resulting PCR product wasdigested with EcoRi and sub-cloned into the MAPS1A-M15-pET plasmid. Theligated products were then transformed into E. coli and transformantswith the correct insert and orientation were identified by restrictiondigest and verified by DNA sequencing. The expressed recombinant proteinwas purified by affinity chromatography over a Ni column. The DNA andamino acid sequences of the fusion protein MAPS1A-M15-LbeIF4A areprovided in SEQ ID NO: 94 and 95, respectively.

[0249] Additional fusion proteins were prepared using the methodologydescribed above. The amino acid sequences for the fusion proteinsMAPS1A-M15-Lbhsp83 and MAPS1A-M15-Lbhsp83-LeIF4A are provided in SEQ IDNO: 96 and 97, respectively. The DNA sequence that encodes the aminoacid sequence of SEQ ID NO: 97 is provided in SEQ ID NO: 98. The DNAsequences of MAPS1A-M15-Lbhsp83 and MAPSlA-Mi5-Lbhsp83-LeIF4A vectorsemployed in DNA vaccines are provided in SEQ ID NO: 99 and 100,respectively.

Example 20 Use of Leishmania Fusion Proteins Plus Adjuvant forVaccination against Leishmania Infection

[0250] The ability of the Leishmania fusion proteins MAPS1A-M15(referred to as the diFusion) and MAPS1A-M15-LbeIF4A (referred to as thetriFusion), plus adjuvant, to confer protection against disease in theexperimental murine leishmaniasis model system was examined as follows.

[0251] The diFusion and triFusion were prepared as described above. In afirst series of experiments, groups of BALB/c mice were immunized witheither the individual recombinant antigens, (MAPS1A, M15 or LbeIF4A),the diFusion or the triFusion, with IL-12 as an adjuvant, as describedabove in Example 17. Control mice were immunized with IL-12 alone orsaline. Before challenge, some mice (three per group) were sacrificedand the immune responses to the fusion proteins and to the individualantigens were investigated. Both T cell (cytokine production by spleencells) and B cell responses (antibody response) were evaluated. Theresults indicated that immunization of mice with the fusion proteins didnot interfere with the immunogenicity of the individual antigens. Morespecifically, Th1 responses (namely induction of IFN-γ production andspecific IgG2a production) were observed to both MAPS1A and M15, whenmice were immunized with both the diFusion and triFusion recombinantproteins. In addition, immunization with the triFusion resulted in goodimmune response to LeIF.

[0252] To evaluate the protection conferred by these fusion proteins,the immunized and control mice were infected in the right footpad with2×10³ amastigote forms of L. major and footpad swelling was measuredweekly thereafter. The results, shown in FIG. 33, clearly indicated thatboth fusion proteins induced protection comparable to MAPS1A and M15.

[0253] A second series of experiments was performed in which MPL-SE(Ribi ImmunoChem Research Inc. (Hamilton, Mont.) was employed as theadjuvant. BALB/c mice were immunized three times (three weeks interval)with 2 μg of the individual antigens (MAPS1A, M15 or LbeIF4A), diFusionor triFusion proteins plus MPL-SE, and tested for immunogenicity of theantigens and for protection as described above. As with the experimentsperformed with IL-12 as adjuvant, the mice immunized with the individualantigens as well as with the fusion proteins showed both specific T andB cell responses to the immunizing antigens. Moreover, no antigencompetition between the individual antigens was observed when the fusionproteins were used as immunogens.

[0254] As with the protection studies in which IL-12 was used asadjuvant, protection was achieved with the individual antigens MAS1A andM15, as well as with the two fusion proteins (FIG. 34). Slightly betterprotection was observed in the group of mice immunized with thetriFusion than in mice immunized with the diFusion.

Example 21 Formulation of Compositions Comprising Leishmania FusionProteins

[0255] A stable preparation of the tri-fusion of MAPS1A, M15 and LbeIF4Adescribed above was prepared as follows. The purified protein was putinto ammonium bicarbonate buffer (pH 8.0) by dialysis, and the followingwere added: 5% (w/v) mannitol, sucrose at 10:1 (w/w) excess sucrose toprotein and 0.1% (v/v) polysorbate 80. The protein was lyophilized todryness to yield a stable powder which can be readily resuspended asneeded.

[0256] From the foregoing, it will be appreciated that, althoughspecific embodiments of the invention have been described herein for thepurpose of illustration, various modifications may be made withoutdeviating from the spirit and scope of the invention.

0 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 122 <210> SEQ ID NO 1<211> LENGTH: 3134 <212> TYPE: DNA <213> ORGANISM: Leishmania major<220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (1)...(3134)<223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 1 caagtgtcgaaggacagtgt tcnccgtgtg agatcgccgg ctgtgcgtgt gaaggcggtg 60 ccatcgganaaacaacaccg gtgganccgc aggaaaccat ctttctccgc aggtctcttt 120 ttgttgtcgattgagagtgc nccaaaccct gctggtgccc ttctcacata tcatgttttt 180 cgttgtgcgctcgctttgcc tttcctctcc tttccctctc ttccgtggtg ccgtgtatac 240 ttctggcacccgctacgtca cttcgctggt ttgaacagaa ccactgtgaa cacccacggg 300 cgatcgcacacatacacatc cctcactcac acacacagct acatctatcc tacataaagc 360 tgaaaaaaaagtctacgaac aattttgttt ttacagtgcg ttgccgcaca tttctccgta 420 atggacgcaactgagctgaa gaacaagggg aacgaagagt tctccgccgg ccgctatgtg 480 gaggcggtgaactacttctc aaaggcgatc cagttggatg agcagaacag tgtcctctac 540 agcaaccgctccgcctgttt tgcagccatg cagaaataca aggacgcgct ggacgacgcc 600 gacaagtgcatctcgatcaa gccgaattgg gccaagggct acgtgcgccg aggagcagct 660 ctccatggcatgcgccgcta cgacgatgcc attgccgcgt atgaaaaggg gctcaaggtg 720 gacccttccaacagcggctg cgcgcagggc gtgaaggacg tgcaggtagc caaggcccgc 780 gaagcacgtgaccccatcgc tcgcgtcttc accccggagg cgttccgcaa gatccaagag 840 aatcccaagctgtctctact tatgctgcag ccggactacg tgaagatggt agacaccgtc 900 atccgcgacccttcgcaggg ccggctgtac atggaagacc agcgctttgc cctgacgctc 960 atgtacctgagcggaatgaa gattcccaac gatggtgatg gcgaggagga ggaacgtccg 1020 tctgcgaaggcggcagagac agcgaagcca aaagaggaga agcctctcac cgacaacgag 1080 aaggaggccctggcgctcaa ggaggagggc aacaagctgt acctctcgaa gaagtttgag 1140 gaggcgctgaccaagtacca agaggcgcag gtgaaagacc ccaacaacac tttatacatt 1200 ctgaacgtgtcggccgtgta cttcgagcag ggtgactacg acaagtgcat cgccgagtgc 1260 gagcacggtatcgagcacgg tcgcgagaac cactgcgact acacaatcat tgcgaagctc 1320 atgacccggaacgccttgtg cctccagagg cagaggaagt acgaggctgc tatcgacctt 1380 tacaagcgcgcccttgtcga gtggcgtaac cctgacaccc tcaagaagct gacggagtgc 1440 gagaaggagcaccaaaaggc ggtggaggaa gcctacatcg atcctgagat cgcgaagcag 1500 aagaaagacgaaggtaacca gtacttcaag gaggataagt tccccgaggc cgtggcagcg 1560 tacacggaggccatcaagcg caaccctgcc gagcacacct cctacagcaa tcgcgcggcc 1620 gcgtacatcaagcttggagc cttcaacgac gccctcaagg acgcggagaa gtgcattgag 1680 ctgaagcccgactttgttaa gggctacgcg cgcaagggtc atgcttactt ttggaccaag 1740 cagtacaaccgcgcgctgca ggcgtacgat gagggcctca aggtggaccc gagcaatgcg 1800 gactgcaaggatgggcggta tcgcacaatc atgaagattc aggagatggc atctggccaa 1860 tccgcggatggcgacgaggc ggcgcgccgg gccatggacg atcctgaaat cgcggcaatc 1920 atgcaagatagctacatgca actagtgttg aaggagatgc agaacgatcc cacgcgcatt 1980 caggagtacatgaaggactc cgggatctca tcgaagatca acaagctgat ttcagctggc 2040 atcattcgttttggtcagta gacttctacg ctgcctcatc ttttccgtgt ctttgcgtcg 2100 gcgggtatcgtaaagcacaa taaagcagcg attcacatgc acgagtaaag tgctgcgcct 2160 ctcaaacacgacgtcgaggc tgtggtgcag atgcgcgtcc tgcatgaagg tagtgaagag 2220 gaaagtaagggatgttgttt gtgggccttc gtggctgcgc acacacctct tatctccttc 2280 gcttggtaccttctcccttt ttcgtcttca cccccctttc tcttctcacg ctctccctgg 2340 cgcggtggtgcaacgatttc gttttattta cgtctgtgta gctcctctat tcaacggtgc 2400 gatgacgctaacgaagctgg cctgtattcg gctaaggcga aggcaaaaga ctaggagggg 2460 ggggggaaggagacggcgtg accatcactg cgaagaaaca agccgaagaa aaggccccga 2520 acgcctgcatttccgcgcgc cctcgcccgc cttccttcct tccttcgctc tctctctctc 2580 tctctctcgctatcttctca acggagacat gaaaggcgtt tgttaggaaa agaggggggg 2640 gggaagagtgggacgacgcg ctgcgtcttt tgggcactgg tcacgtgcgt caccctcttt 2700 ttttatctctattggcactg tcttgtttct tttccctttc ctatcatacg cgtctcgcaa 2760 acgactccgcgctgagcagc catgtgctgc ggcgtggagg aagtacacag acatcacgga 2820 tgcatatgtgcgcgtccgtg tacgcgcttg tatggggctt ctaacagcgc ctgtgtgtgt 2880 ttgtgtgtgtgtgtgtgtgt gtgtctgtgt atttcgagcg tctgtatgct attctattaa 2940 gcaccgaagaagagacacac acgacagcga aggagatggt gtcggctttt cggctaatca 3000 ctcccttccatagcttctct gaaggaggct ctcttccaga ggaatagact gcagatgggg 3060 tccacgtttatctgaggagt caacggaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3120 aaaaaaaactcgag 3134 <210> SEQ ID NO 2 <211> LENGTH: 546 <212> TYPE: PRT <213>ORGANISM: Leishmania major <400> SEQUENCE: 2 Met Asp Ala Thr Glu Leu LysAsn Lys Gly Asn Glu Glu Phe Ser Ala 1 5 10 15 Gly Arg Tyr Val Glu AlaVal Asn Tyr Phe Ser Lys Ala Ile Gln Leu 20 25 30 Asp Glu Gln Asn Ser ValLeu Tyr Ser Asn Arg Ser Ala Cys Phe Ala 35 40 45 Ala Met Gln Lys Tyr LysAsp Ala Leu Asp Asp Ala Asp Lys Cys Ile 50 55 60 Ser Ile Lys Pro Asn TrpAla Lys Gly Tyr Val Arg Arg Gly Ala Ala 65 70 75 80 Leu His Gly Met ArgArg Tyr Asp Asp Ala Ile Ala Ala Tyr Glu Lys 85 90 95 Gly Leu Lys Val AspPro Ser Asn Ser Gly Cys Ala Gln Gly Val Lys 100 105 110 Asp Val Gln ValAla Lys Ala Arg Glu Ala Arg Asp Pro Ile Ala Arg 115 120 125 Val Phe ThrPro Glu Ala Phe Arg Lys Ile Gln Glu Asn Pro Lys Leu 130 135 140 Ser LeuLeu Met Leu Gln Pro Asp Tyr Val Lys Met Val Asp Thr Val 145 150 155 160Ile Arg Asp Pro Ser Gln Gly Arg Leu Tyr Met Glu Asp Gln Arg Phe 165 170175 Ala Leu Thr Leu Met Tyr Leu Ser Gly Met Lys Ile Pro Asn Asp Gly 180185 190 Asp Gly Glu Glu Glu Glu Arg Pro Ser Ala Lys Ala Ala Glu Thr Ala195 200 205 Lys Pro Lys Glu Glu Lys Pro Leu Thr Asp Asn Glu Lys Glu AlaLeu 210 215 220 Ala Leu Lys Glu Glu Gly Asn Lys Leu Tyr Leu Ser Lys LysPhe Glu 225 230 235 240 Glu Ala Leu Thr Lys Tyr Gln Glu Ala Gln Val LysAsp Pro Asn Asn 245 250 255 Thr Leu Tyr Ile Leu Asn Val Ser Ala Val TyrPhe Glu Gln Gly Asp 260 265 270 Tyr Asp Lys Cys Ile Ala Glu Cys Glu HisGly Ile Glu His Gly Arg 275 280 285 Glu Asn His Cys Asp Tyr Thr Ile IleAla Lys Leu Met Thr Arg Asn 290 295 300 Ala Leu Cys Leu Gln Arg Gln ArgLys Tyr Glu Ala Ala Ile Asp Leu 305 310 315 320 Tyr Lys Arg Ala Leu ValGlu Trp Arg Asn Pro Asp Thr Leu Lys Lys 325 330 335 Leu Thr Glu Cys GluLys Glu His Gln Lys Ala Val Glu Glu Ala Tyr 340 345 350 Ile Asp Pro GluIle Ala Lys Gln Lys Lys Asp Glu Gly Asn Gln Tyr 355 360 365 Phe Lys GluAsp Lys Phe Pro Glu Ala Val Ala Ala Tyr Thr Glu Ala 370 375 380 Ile LysArg Asn Pro Ala Glu His Thr Ser Tyr Ser Asn Arg Ala Ala 385 390 395 400Ala Tyr Ile Lys Leu Gly Ala Phe Asn Asp Ala Leu Lys Asp Ala Glu 405 410415 Lys Cys Ile Glu Leu Lys Pro Asp Phe Val Lys Gly Tyr Ala Arg Lys 420425 430 Gly His Ala Tyr Phe Trp Thr Lys Gln Tyr Asn Arg Ala Leu Gln Ala435 440 445 Tyr Asp Glu Gly Leu Lys Val Asp Pro Ser Asn Ala Asp Cys LysAsp 450 455 460 Gly Arg Tyr Arg Thr Ile Met Lys Ile Gln Glu Met Ala SerGly Gln 465 470 475 480 Ser Ala Asp Gly Asp Glu Ala Ala Arg Arg Ala MetAsp Asp Pro Glu 485 490 495 Ile Ala Ala Ile Met Gln Asp Ser Tyr Met GlnLeu Val Leu Lys Glu 500 505 510 Met Gln Asn Asp Pro Thr Arg Ile Gln GluTyr Met Lys Asp Ser Gly 515 520 525 Ile Ser Ser Lys Ile Asn Lys Leu IleSer Ala Gly Ile Ile Arg Phe 530 535 540 Gly Gln 545 <210> SEQ ID NO 3<211> LENGTH: 676 <212> TYPE: DNA <213> ORGANISM: Leishmania donovani<400> SEQUENCE: 3 aattcggcac gaggcattgt gcataatggt caagtcccac tacatctgcgcgggccgcct 60 ggtgcgcatc ctgcgtggcc cccgccagga ccgcgttggt gtgatcgtcgacattgtcga 120 cgcgaaccgc gtgctggtgg agaacccgga ggacgcgaag atgtggcgccacgtgcagaa 180 cctgaagaac gtggagccgc tgaagtactg cgtgagcgtc agccgcaactgcagcgcgaa 240 ggcgctgaag gatgcgctgg cctcgtcgaa ggcgctggag aagtacgcgaagacgcgcac 300 tgctgcgcgc gtggaggcga agaaggcgtg cgccgcgtcg acggacttcgagcgctacca 360 gctgcgcgtt gcgcgccgtt ctcgcgcgca ctgggcgcgc aaggtgttcgacgagaagga 420 cgcgaagacg cccgtgtcgt ggcacaaggt tgcgctgaag aagatgcagaagaaggccgc 480 aaagatggac tcgaccgagg gcgctaagag gcgcatgcag aaggcgatcgctgcccgcaa 540 ggcgaaaaag taaggccata ccctcacttc gcttgtttcg tgatttttcgtgggagtcgg 600 tggccctacc agcggtcttt cattggctta tttctatccg gtctgaaagaggtacaaaaa 660 aaaaaaaaaa aaaaaa 676 <210> SEQ ID NO 4 <211> LENGTH: 175<212> TYPE: PRT <213> ORGANISM: Leishmania donovani <400> SEQUENCE: 4Met Val Lys Ser His Tyr Ile Cys Ala Gly Arg Leu Val Arg Ile Leu 1 5 1015 Arg Gly Pro Arg Gln Asp Arg Val Gly Val Ile Val Asp Ile Val Asp 20 2530 Ala Asn Arg Val Leu Val Glu Asn Pro Glu Asp Ala Lys Met Trp Arg 35 4045 His Val Gln Asn Leu Lys Asn Val Glu Pro Leu Lys Tyr Cys Val Ser 50 5560 Val Ser Arg Asn Cys Ser Ala Lys Ala Leu Lys Asp Ala Leu Ala Ser 65 7075 80 Ser Lys Ala Leu Glu Lys Tyr Ala Lys Thr Arg Thr Ala Ala Arg Val 8590 95 Glu Ala Lys Lys Ala Cys Ala Ala Ser Thr Asp Phe Glu Arg Tyr Gln100 105 110 Leu Arg Val Ala Arg Arg Ser Arg Ala His Trp Ala Arg Lys ValPhe 115 120 125 Asp Glu Lys Asp Ala Lys Thr Pro Val Ser Trp His Lys ValAla Leu 130 135 140 Lys Lys Met Gln Lys Lys Ala Ala Lys Met Asp Ser ThrGlu Gly Ala 145 150 155 160 Lys Arg Arg Met Gln Lys Ala Ile Ala Ala ArgLys Ala Lys Lys 165 170 175 <210> SEQ ID NO 5 <211> LENGTH: 2040 <212>TYPE: DNA <213> ORGANISM: Leishmania braziliensis <400> SEQUENCE: 5cgcggtggcg gccgctctag aactagtgga tcccccgggc tgcaggaatt cggcacgaga 60gagcctgacg gacccggcgg tgctgggcga ggagactcac ctgcgcgtcc gcgtggtgcc 120ggacaaggcg aacaagacgc tgacggtgga ggataacggc atcggcatga ccaaggcgga 180cctcgtgaac aatctgggca cgatcgcgcg ctccggcacg aaggctttca tggaggcact 240ggaggccggc ggcgacatga gcatgatcgg ccagttcggt gtcggcttct actccgcgta 300ccttgtggcg gaccgcgtga cggtggtgtc gaagaacaac tcggacgagg cgtactggga 360atcgtctgcg gggggcacgt tcaccatcac gagcgtgcag gagtcggaca tgaagcgcgg 420cacgagtaca acgctgcacc taaaggagga ccagcaggag tacctggagg agcgccgggt 480gaaggagctg atcaagaagc actccgagtt catcggctac gacatcgagc tgatggtgga 540gaagacggcg gagaaggagg tgacggacga ggacgaggag gaggacgagt cgaagaagaa 600gtcctgcggg gacgagggcg agccgaaggt ggaggaggtg acggagggcg gcgaggacaa 660gaagaagaag acgaagaagg tgaaggaggt gaagaagacg tacgaggtca agaacaagca 720caagccgctc tggacgcgcg acacgaagga cgtgacgaag gaggagtacg cggccttcta 780caaggccatc tccaacgact gggaggacac ggcggcgacg aagcacttct cggtggaggg 840ccagctggag ttccgcgcga tcgcgttcgt gccgaagcgc gcgccgttcg acatgttcga 900gccgaacaag aagcgcaaca acatcaagct gtacgtgcgc cgcgtgttca tcatggacaa 960ctgcgaggac ctgtgcccgg actggctcgg cttcgtgaag ggcgtcgtgg acagcgagga 1020cctgccgctg aacatctcgc gcgagaacct gcagcagaac aagatcctga aggtgatccg 1080caagaacatc gtgaagaagt gcctggagct gttcgaagag atagcggaga acaaggagga 1140ctacaagcag ttctacgagc agttcggcaa gaacatcaag ctgggcatcc acgaggacac 1200ggcgaaccgc aagaagctga tggagttgct gcgcttctac agcaccgagt cgggggagga 1260gatgacgaca ctgaaggact acgtgacgcg catgaagccg gagcagaagt cgatctacta 1320catcactggc gacagcaaga agaagctgga gtcgtcgccg ttcatcgaga aggcgagacg 1380ctgcgggctc gaggtgctgt tcatgacgga gccgatcgac gagtacgtga tgcagcaggt 1440gaaggacttc gaggacaaga agttcgcgtg cctgacgaag gaaggcgtgc acttcgagga 1500gtccgaggag gagaagaagc agcgcgagga gaagaaggcg gcgtgcgaga agctgtgcaa 1560gacgatgaag gaggtgctgg gcgacaaggt ggagaaggtg accgtgtcgg agcgcctgtt 1620gacgtcgccg tgcatcctgg tgacgtcgga gtttgggtgg tcggcgcaca tggaacagat 1680catgcgcaac caggcgctgc gcgactccag catggcgcag tacatggtgt ccaagaagac 1740gatggaggtg aaccccgacc accccatcat caaggagctg cgccgccgcg tggaggcgga 1800cgagaacgac aaggccgtga aggacctcgt cttcctgctc ttcgacacgt cgctgctcac 1860gtccggcttc cagctggatg accccaccgg ctacgccgag cgcatcaacc gcatgatcaa 1920gctcggcctg tcgctcgacg aggaggagga ggaggtcgcc gaggcgccgc cggccgaggc 1980agcccccgcg gaggtcaccg ccggcacctc cagcatggag caggtggact gagccggtaa 2040<210> SEQ ID NO 6 <211> LENGTH: 656 <212> TYPE: PRT <213> ORGANISM:Leshmania brailiensis <400> SEQUENCE: 6 Ser Leu Thr Asp Pro Ala Val LeuGly Glu Glu Thr His Leu Arg Val 1 5 10 15 Arg Val Val Pro Asp Lys AlaAsn Lys Thr Leu Thr Val Glu Asp Asn 20 25 30 Gly Ile Gly Met Thr Lys AlaAsp Leu Val Asn Asn Leu Gly Thr Ile 35 40 45 Ala Arg Ser Gly Thr Lys AlaPhe Met Glu Ala Leu Glu Ala Gly Gly 50 55 60 Asp Met Ser Met Ile Gly GlnPhe Gly Val Gly Phe Tyr Ser Ala Tyr 65 70 75 80 Leu Val Ala Asp Arg ValThr Val Val Ser Lys Asn Asn Ser Asp Glu 85 90 95 Ala Tyr Trp Glu Ser SerAla Gly Gly Thr Phe Thr Ile Thr Ser Val 100 105 110 Gln Glu Ser Asp MetLys Arg Gly Thr Ser Thr Thr Leu His Leu Lys 115 120 125 Glu Asp Gln GlnGlu Tyr Leu Glu Glu Arg Arg Val Lys Glu Leu Ile 130 135 140 Lys Lys HisSer Glu Phe Ile Gly Tyr Asp Ile Glu Leu Met Val Glu 145 150 155 160 LysThr Ala Glu Lys Glu Val Thr Asp Glu Asp Glu Glu Glu Asp Glu 165 170 175Ser Lys Lys Lys Ser Cys Gly Asp Glu Gly Glu Pro Lys Val Glu Glu 180 185190 Val Thr Glu Gly Gly Glu Asp Lys Lys Lys Lys Thr Lys Lys Val Lys 195200 205 Glu Val Lys Lys Thr Tyr Glu Val Lys Asn Lys His Lys Pro Leu Trp210 215 220 Thr Arg Asp Thr Lys Asp Val Thr Lys Glu Glu Tyr Ala Ala PheTyr 225 230 235 240 Lys Ala Ile Ser Asn Asp Trp Glu Asp Thr Ala Ala ThrLys His Phe 245 250 255 Ser Val Glu Gly Gln Leu Glu Phe Arg Ala Ile AlaPhe Val Pro Lys 260 265 270 Arg Ala Pro Phe Asp Met Phe Glu Pro Asn LysLys Arg Asn Asn Ile 275 280 285 Lys Leu Tyr Val Arg Arg Val Phe Ile MetAsp Asn Cys Glu Asp Leu 290 295 300 Cys Pro Asp Trp Leu Gly Phe Val LysGly Val Val Asp Ser Glu Asp 305 310 315 320 Leu Pro Leu Asn Ile Ser ArgGlu Asn Leu Gln Gln Asn Lys Ile Leu 325 330 335 Lys Val Ile Arg Lys AsnIle Val Lys Lys Cys Leu Glu Leu Phe Glu 340 345 350 Glu Ile Ala Glu AsnLys Glu Asp Tyr Lys Gln Phe Tyr Glu Gln Phe 355 360 365 Gly Lys Asn IleLys Leu Gly Ile His Glu Asp Thr Ala Asn Arg Lys 370 375 380 Lys Leu MetGlu Leu Leu Arg Phe Tyr Ser Thr Glu Ser Gly Glu Glu 385 390 395 400 MetThr Thr Leu Lys Asp Tyr Val Thr Arg Met Lys Pro Glu Gln Lys 405 410 415Ser Ile Tyr Tyr Ile Thr Gly Asp Ser Lys Lys Lys Leu Glu Ser Ser 420 425430 Pro Phe Ile Glu Lys Ala Arg Arg Cys Gly Leu Glu Val Leu Phe Met 435440 445 Thr Glu Pro Ile Asp Glu Tyr Val Met Gln Gln Val Lys Asp Phe Glu450 455 460 Asp Lys Lys Phe Ala Cys Leu Thr Lys Glu Gly Val His Phe GluGlu 465 470 475 480 Ser Glu Glu Glu Lys Lys Gln Arg Glu Glu Lys Lys AlaAla Cys Glu 485 490 495 Lys Leu Cys Lys Thr Met Lys Glu Val Leu Gly AspLys Val Glu Lys 500 505 510 Val Thr Val Ser Glu Arg Leu Leu Thr Ser ProCys Ile Leu Val Thr 515 520 525 Ser Glu Phe Gly Trp Ser Ala His Met GluGln Ile Met Arg Asn Gln 530 535 540 Ala Leu Arg Asp Ser Ser Met Ala GlnTyr Met Val Ser Lys Lys Thr 545 550 555 560 Met Glu Val Asn Pro Asp HisPro Ile Ile Lys Glu Leu Arg Arg Arg 565 570 575 Val Glu Ala Asp Glu AsnAsp Lys Ala Val Lys Asp Leu Val Phe Leu 580 585 590 Leu Phe Asp Thr SerLeu Leu Thr Ser Gly Phe Gln Leu Asp Asp Pro 595 600 605 Thr Gly Tyr AlaGlu Arg Ile Asn Arg Met Ile Lys Leu Gly Leu Ser 610 615 620 Leu Asp GluGlu Glu Glu Glu Val Ala Glu Ala Pro Pro Ala Glu Ala 625 630 635 640 AlaPro Ala Glu Val Thr Ala Gly Thr Ser Ser Met Glu Gln Val Asp 645 650 655<210> SEQ ID NO 7 <211> LENGTH: 1771 <212> TYPE: DNA <213> ORGANISM:Leishmania tropica <400> SEQUENCE: 7 caggcccgcg tccaggccct cgaggaggcagcgcgtctcc gcgcggagct ggaggcggcc 60 gaggaggcgg cccgcctgga tgtcatgcatgcggccgagc aggcccgtgt ccaggccctc 120 gaggaggcag cgcgtctccg cgcggagctggaggaggccg aggaggcggc ccgcctggat 180 gtcatgcatg cggccgagca ggcccgcgtccaggccctcg aggaggcagc gcgtctccgc 240 gcggagctgg aggctgccga ggaggcggcgcgcctggagg ccatgcacga ggccgagcag 300 gcccgctccc aggccctcga ggaggcagcgcgtctccgcg cggagctgga ggaagccgag 360 gaggcggccc gcctggatgt catgcatgcggccgagcagg cccgcgtcca ggccctcgag 420 gaggcagcgc gtctccgcgc ggagctggaggaggccgagg aggcggcccg cctggaggcc 480 atgcacgagg ccgagcaggc ccgctcccaggccctcgagg aggcagcgcg tctccgcgcg 540 gagctggagg cggccgagga ggcggcccgcctggatgtca tgcacgaggc cgagcaggcc 600 cgtgtccagg ccctcgagga ggcggcgcgcctggatgtca tgcacgaggc cgagcaggcc 660 cgcgtccagg ccctcgagga ggcagcgcgtctccgcgcgg agctggaggc ggccgaggag 720 gcggcccgcc tggatgtcat gcacgaggccgagcaggccc gcgtccaggc cctcgaggag 780 gcagcgcgtc tccgcgcgga gctggaggcggccgaggagg cggcccgcct ggatgtcatg 840 cacgagggcg agcaggcccg tgtccaggccctcgaggagg cggcccgcct ggaggccatg 900 cacgaggccg agcaggcccg ctcccaggccctcgaggagg cagcgcgtct ctgcgcggag 960 ctggaggctg aggaggagga aaaagatgagcggccggcga cgtcgagcta cagcgaggag 1020 tgcaaagggc gactgctatc gagggcgcggccggatccgc ggaggccgct gccgcggccg 1080 ttcattggga tgtcactgtt ggaggatgtggagaagagta ttctcattgt ggacgggctc 1140 tacagggatg ggccggcgta ccagacgggcatccgcctcg gggatgtcct cttgcgtatc 1200 gcgggggttt acgtggattc aatagcgaaggcgaggcagg tggtcgatgc gcgttgccgc 1260 tgcggctgcg tcgttcccgt gacgctggcgacgaagatga accagcagta cagcgtggct 1320 ctgtatatca tgacggtgga tccgcagcacaacgacaagc cctttttttt tgatgtgcac 1380 atccaccacc gcatcgagag ctcgcacatggggaagaagg cgcagtggat ggaagttctt 1440 gagagcccat ccgtatcttc ggctgccaccacccctctcg tgccgctctt gcgtgagccg 1500 acgccgcgta ggggctcaga gctgcagtcaagtgctcgtt ccgccttcgt tgccacgtct 1560 tacttctcga gcgcgcgcag gtcggtcagctcagaaagtg agcgaccgcg cgggtcctct 1620 agcgtggcta tggcggagga ggcgatcgcgctggcgccgc aagggtatac cccacccaac 1680 caagtgcgcg gccgtagttg acgtctctgtgtgagtgtgt gtcgctccgt ctccttcctt 1740 tttcgtcatg tgttttattc atttcttttt c1771 <210> SEQ ID NO 8 <211> LENGTH: 566 <212> TYPE: PRT <213> ORGANISM:Leishmania tropica <400> SEQUENCE: 8 Gln Ala Arg Val Gln Ala Leu Glu GluAla Ala Arg Leu Arg Ala Glu 1 5 10 15 Leu Glu Ala Ala Glu Glu Ala AlaArg Leu Asp Val Met His Ala Ala 20 25 30 Glu Gln Ala Arg Val Gln Ala LeuGlu Glu Ala Ala Arg Leu Arg Ala 35 40 45 Glu Leu Glu Glu Ala Glu Glu AlaAla Arg Leu Asp Val Met His Ala 50 55 60 Ala Glu Gln Ala Arg Val Gln AlaLeu Glu Glu Ala Ala Arg Leu Arg 65 70 75 80 Ala Glu Leu Glu Ala Ala GluGlu Ala Ala Arg Leu Glu Ala Met His 85 90 95 Glu Ala Glu Gln Ala Arg SerGln Ala Leu Glu Glu Ala Ala Arg Leu 100 105 110 Arg Ala Glu Leu Glu GluAla Glu Glu Ala Ala Arg Leu Asp Val Met 115 120 125 His Ala Ala Glu GlnAla Arg Val Gln Ala Leu Glu Glu Ala Ala Arg 130 135 140 Leu Arg Ala GluLeu Glu Glu Ala Glu Glu Ala Ala Arg Leu Glu Ala 145 150 155 160 Met HisGlu Ala Glu Gln Ala Arg Ser Gln Ala Leu Glu Glu Ala Ala 165 170 175 ArgLeu Arg Ala Glu Leu Glu Ala Ala Glu Glu Ala Ala Arg Leu Asp 180 185 190Val Met His Glu Ala Glu Gln Ala Arg Val Gln Ala Leu Glu Glu Ala 195 200205 Ala Arg Leu Asp Val Met His Glu Ala Glu Gln Ala Arg Val Gln Ala 210215 220 Leu Glu Glu Ala Ala Arg Leu Arg Ala Glu Leu Glu Ala Ala Glu Glu225 230 235 240 Ala Ala Arg Leu Asp Val Met His Glu Ala Glu Gln Ala ArgVal Gln 245 250 255 Ala Leu Glu Glu Ala Ala Arg Leu Arg Ala Glu Leu GluAla Ala Glu 260 265 270 Glu Ala Ala Arg Leu Asp Val Met His Glu Gly GluGln Ala Arg Val 275 280 285 Gln Ala Leu Glu Glu Ala Ala Arg Leu Glu AlaMet His Glu Ala Glu 290 295 300 Gln Ala Arg Ser Gln Ala Leu Glu Glu AlaAla Arg Leu Cys Ala Glu 305 310 315 320 Leu Glu Ala Glu Glu Glu Glu LysAsp Glu Arg Pro Ala Thr Ser Ser 325 330 335 Tyr Ser Glu Glu Cys Lys GlyArg Leu Leu Ser Arg Ala Arg Pro Asp 340 345 350 Pro Arg Arg Pro Leu ProArg Pro Phe Ile Gly Met Ser Leu Leu Glu 355 360 365 Asp Val Glu Lys SerIle Leu Ile Val Asp Gly Leu Tyr Arg Asp Gly 370 375 380 Pro Ala Tyr GlnThr Gly Ile Arg Leu Gly Asp Val Leu Leu Arg Ile 385 390 395 400 Ala GlyVal Tyr Val Asp Ser Ile Ala Lys Ala Arg Gln Val Val Asp 405 410 415 AlaArg Cys Arg Cys Gly Cys Val Val Pro Val Thr Leu Ala Thr Lys 420 425 430Met Asn Gln Gln Tyr Ser Val Ala Leu Tyr Ile Met Thr Val Asp Pro 435 440445 Gln His Asn Asp Lys Pro Phe Phe Phe Asp Val His Ile His His Arg 450455 460 Ile Glu Ser Ser His Met Gly Lys Lys Ala Gln Trp Met Glu Val Leu465 470 475 480 Glu Ser Pro Ser Val Ser Ser Ala Ala Thr Thr Pro Leu ValPro Leu 485 490 495 Leu Arg Glu Pro Thr Pro Arg Arg Gly Ser Glu Leu GlnSer Ser Ala 500 505 510 Arg Ser Ala Phe Val Ala Thr Ser Tyr Phe Ser SerAla Arg Arg Ser 515 520 525 Val Ser Ser Glu Ser Glu Arg Pro Arg Gly SerSer Ser Val Ala Met 530 535 540 Ala Glu Glu Ala Ile Ala Leu Ala Pro GlnGly Tyr Thr Pro Pro Asn 545 550 555 560 Gln Val Arg Gly Arg Ser 565<210> SEQ ID NO 9 <211> LENGTH: 1618 <212> TYPE: DNA <213> ORGANISM:Leishmania braziliensis <400> SEQUENCE: 9 ccactctctc ggtcgtctgtctcccacgcg cgcacgcagt tgatttccgc cttcttaaac 60 gctctctttt tttttatttttcacctgacc aaccgcacca cgtcggcctc catcatgtcg 120 cagcaagacc gagttgccccacaggaccag gactcgttcc tcgacgacca gcccggcgtc 180 cgcccgatcc cgtccttcgatgacatgccg ttgcaccaga accttctgcg cggcatctac 240 tcgtacggct tcgagaaaccgtccagcatc cagcagcgcg ccatcgcccc cttcacgcgc 300 ggcggcgaca tcatcgcgcaggcgcagtcc ggtaccggca agacgggcgc cttctccatc 360 ggcctgctgc agcgcctggacttccgccac aacctgatcc agggcctcgt gctctccccg 420 acccgcgagc tggccctgcagacggcggag gtgatcagcc gcatcggcga gttcctgtcg 480 aacagcgcga agttctgtgagacctttgtg ggtggcacgc gcgtgcagga tgacctgcgc 540 aagctgcagg ctggcgtcgtcgtcgccgtg gggacgccgg gccgcgtgtc cgacgtgatc 600 aagcgcggcg cgctgcgcaccgagtccctg cgcgtgctgg tgctcgacga ggctgatgag 660 atgctgtctc agggcttcgcggatcagatt tacgagatct tccgcttcct gccgaaggac 720 atccaggtcg cgctcttctccgccacgatg ccggaggagg tgctggagct gacaaagaag 780 ttcatgcgcg accccgtacgcattctcgtg aagcgcgaga gcctgacgct ggagggcatc 840 aagcagttct tcatcgccgtcgaggaggag cacaagctgg acacgctgat ggacctgtac 900 gagaccgtgt ccatcgcgcagtccgtcatc ttcgccaaca cccgccgcaa ggtggactgg 960 atcgccgaga agctgaatcagagcaaccac accgtcagca gcatgcacgc cgagatgccc 1020 aagagcgacc gcgagcgcgtcatgaacacc ttccgcagcg gcagctcccg cgtgctcgta 1080 acgaccgacc tcgtggcccgcggcatcgac gtgcaccacg tgaacatcgt catcaacttc 1140 gacctgccga cgaacaaggagaactacctg caccgcattg gccgcggcgg ccgctacggc 1200 gtaaagggtg ttgccatcaacttcgtgacg gagaaagacg tggagctgct gcacgagatc 1260 gaggggcact accacacgcagatcgatgag ctcccggtgg actttgccgc ctacctcggc 1320 gagtgagcgg gcccctgccccccttccctg cccccctctc gcgacgagag aacgcacatc 1380 gtaacacagc cacgcgaacgatagtaaggg cgtgcggcgg cgttcccctc ctcctgccag 1440 cggcccccct ccgcagcgcttctcttttga gaggggggca gggggaggcg ctgcgcctgg 1500 ctggatgtgt gcttgagcttgcattccgtc aagcaagtgc tttgttttaa ttatgcgcgc 1560 cgttttgttg ctcgtccctttcgttggtgt tttttcggcc gaaacggcgt ttaaagca 1618 210> SEQ ID NO 10 <211>LENGTH: 403 <212> TYPE: PRT <213> ORGANISM: Leishmania braziliensis<400> SEQUENCE: 10 Met Ser Gln Gln Asp Arg Val Ala Pro Gln Asp Gln AspSer Phe Leu 1 5 10 15 Asp Asp Gln Pro Gly Val Arg Pro Ile Pro Ser PheAsp Asp Met Pro 20 25 30 Leu His Gln Asn Leu Leu Arg Gly Ile Tyr Ser TyrGly Phe Glu Lys 35 40 45 Pro Ser Ser Ile Gln Gln Arg Ala Ile Ala Pro PheThr Arg Gly Gly 50 55 60 Asp Ile Ile Ala Gln Ala Gln Ser Gly Thr Gly LysThr Gly Ala Phe 65 70 75 80 Ser Ile Gly Leu Leu Gln Arg Leu Asp Phe ArgHis Asn Leu Ile Gln 85 90 95 Gly Leu Val Leu Ser Pro Thr Arg Glu Leu AlaLeu Gln Thr Ala Glu 100 105 110 Val Ile Ser Arg Ile Gly Glu Phe Leu SerAsn Ser Ala Lys Phe Cys 115 120 125 Glu Thr Phe Val Gly Gly Thr Arg ValGln Asp Asp Leu Arg Lys Leu 130 135 140 Gln Ala Gly Val Val Val Ala ValGly Thr Pro Gly Arg Val Ser Asp 145 150 155 160 Val Ile Lys Arg Gly AlaLeu Arg Thr Glu Ser Leu Arg Val Leu Val 165 170 175 Leu Asp Glu Ala AspGlu Met Leu Ser Gln Gly Phe Ala Asp Gln Ile 180 185 190 Tyr Glu Ile PheArg Phe Leu Pro Lys Asp Ile Gln Val Ala Leu Phe 195 200 205 Ser Ala ThrMet Pro Glu Glu Val Leu Glu Leu Thr Lys Lys Phe Met 210 215 220 Arg AspPro Val Arg Ile Leu Val Lys Arg Glu Ser Leu Thr Leu Glu 225 230 235 240Gly Ile Lys Gln Phe Phe Ile Ala Val Glu Glu Glu His Lys Leu Asp 245 250255 Thr Leu Met Asp Leu Tyr Glu Thr Val Ser Ile Ala Gln Ser Val Ile 260265 270 Phe Ala Asn Thr Arg Arg Lys Val Asp Trp Ile Ala Glu Lys Leu Asn275 280 285 Gln Ser Asn His Thr Val Ser Ser Met His Ala Glu Met Pro LysSer 290 295 300 Asp Arg Glu Arg Val Met Asn Thr Phe Arg Ser Gly Ser SerArg Val 305 310 315 320 Leu Val Thr Thr Asp Leu Val Ala Arg Gly Ile AspVal His His Val 325 330 335 Asn Ile Val Ile Asn Phe Asp Leu Pro Thr AsnLys Glu Asn Tyr Leu 340 345 350 His Arg Ile Gly Arg Gly Gly Arg Tyr GlyVal Lys Gly Val Ala Ile 355 360 365 Asn Phe Val Thr Glu Lys Asp Val GluLeu Leu His Glu Ile Glu Gly 370 375 380 His Tyr His Thr Gln Ile Asp GluLeu Pro Val Asp Phe Ala Ala Tyr 385 390 395 400 Leu Gly Glu <210> SEQ IDNO 11 <211> LENGTH: 12 <212> TYPE: PRT <213> ORGANISM: Leishmaniadonovani <220> FEATURE: <221> NAME/KEY: VARIANT <222> LOCATION:(1)...(5) <223> OTHER INFORMATION: Xaa = any amino acid <221> NAME/KEY:VARIANT <222> LOCATION: (6)...(6) <223> OTHER INFORMATION: Xaa = Leu orLys <221> NAME/KEY: VARIANT <222> LOCATION: (7)...(12) <223> OTHERINFORMATION: Xaa = any amino acid <400> SEQUENCE: 11 Xaa Gln Xaa Pro GlnXaa Val Phe Asp Glu Xaa Xaa 1 5 10 <210> SEQ ID NO 12 <211> LENGTH: 26<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: Sense PCR pri mer <221> NAME/KEY: modified_base <222>LOCATION: (1)...(26) <223> OTHER INFORMATION: I <221> NAME/KEY:misc_feature <222> LOCATION: (1)...(26) <223> OTHER INFORMATION: n =A,T,C or G <400> SEQUENCE: 12 ggaattcccc ncagctngtn ttcgac 26 <210> SEQID NO 13 <211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Leishmaniadonovani <400> SEQUENCE: 13 Lys Val Phe Asp Glu 1 5 <210> SEQ ID NO 14<211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: PCR primer <400> SEQUENCE: 14ggatccatgg tcaagtccca ctacatctgc 30 <210> SEQ ID NO 15 <211> LENGTH: 33<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: PCR primer <400> SEQUENCE: 15 gaattcagac cggatagaaataagccaatg aaa 33 <210> SEQ ID NO 16 <211> LENGTH: 701 <212> TYPE: PRT<213> ORGANISM: Leishmania amozonensis <400> SEQUENCE: 16 Met Thr GluThr Phe Ala Phe Gln Ala Glu Ile Asn Gln Leu Met Ser 1 5 10 15 Leu IleIle Asn Thr Phe Tyr Ser Asn Lys Glu Ile Phe Leu Arg Asp 20 25 30 Val IleSer Asn Ala Ser Asp Ala Cys Asp Lys Ile Arg Tyr Gln Ser 35 40 45 Leu ThrAsp Pro Ala Val Leu Gly Asp Ala Thr Arg Leu Cys Val Arg 50 55 60 Val ValPro Asp Lys Glu Asn Lys Thr Leu Thr Val Glu Asp Asn Gly 65 70 75 80 IleGly Met Thr Lys Ala Asp Leu Val Asn Asn Leu Gly Thr Ile Ala 85 90 95 ArgSer Gly Thr Lys Ala Phe Met Glu Ala Leu Glu Ala Gly Ala Asp 100 105 110Met Ser Met Ile Gly Gln Phe Gly Val Gly Phe Tyr Ser Ala Tyr Leu 115 120125 Val Ala Asp Arg Val Thr Val Thr Ser Lys Asn Asn Ser Asp Glu Val 130135 140 Tyr Val Trp Glu Ser Ser Ala Gly Gly Thr Phe Thr Ile Thr Ser Ala145 150 155 160 Pro Glu Ser Asp Met Lys Leu Pro Ala Arg Ile Thr Leu HisLeu Lys 165 170 175 Glu Asp Gln Leu Glu Tyr Leu Glu Ala Arg Arg Leu LysGlu Leu Ile 180 185 190 Lys Lys His Ser Glu Phe Ile Gly Tyr Asp Ile GluLeu Met Val Glu 195 200 205 Lys Thr Thr Glu Lys Glu Val Thr Asp Glu AspGlu Glu Glu Ala Lys 210 215 220 Lys Ala Asp Glu Asp Gly Glu Glu Pro LysVal Glu Glu Val Thr Glu 225 230 235 240 Gly Glu Glu Asp Lys Lys Lys LysThr Lys Lys Val Lys Glu Val Thr 245 250 255 Lys Glu Tyr Glu Val Gln AsnLys His Lys Pro Leu Trp Thr Arg Asp 260 265 270 Pro Lys Asp Val Thr LysGlu Glu Tyr Ala Ala Phe Tyr Lys Ala Ile 275 280 285 Ser Asn Asp Trp GluAsp Pro Pro Ala Thr Lys His Phe Ser Val Glu 290 295 300 Gly Gln Leu GluPhe Arg Ala Ile Met Phe Val Pro Lys Arg Ala Pro 305 310 315 320 Phe AspMet Leu Glu Pro Asn Lys Lys Arg Asn Asn Ile Lys Leu Tyr 325 330 335 ValArg Arg Val Phe Ile Met Asp Asn Cys Glu Asp Leu Cys Pro Asp 340 345 350Trp Leu Gly Phe Val Lys Gly Val Val Asp Ser Glu Asp Leu Pro Leu 355 360365 Asn Ile Ser Arg Glu Asn Leu Gln Gln Asn Lys Ile Leu Lys Val Ile 370375 380 Arg Lys Asn Ile Val Lys Lys Cys Leu Glu Met Phe Glu Glu Val Ala385 390 395 400 Glu Asn Lys Glu Asp Tyr Lys Gln Phe Tyr Glu Gln Phe GlyLys Asn 405 410 415 Ile Lys Leu Gly Ile His Glu Asp Thr Ala Asn Arg LysLys Leu Met 420 425 430 Glu Leu Leu Arg Phe Tyr Ser Thr Glu Ser Gly GluVal Met Thr Thr 435 440 445 Leu Lys Asp Tyr Val Thr Arg Met Lys Ala GluGln Asn Ser Ile Tyr 450 455 460 Tyr Ile Thr Gly Asp Ser Lys Lys Lys LeuGlu Ser Ser Pro Phe Ile 465 470 475 480 Glu Gln Ala Lys Arg Arg Gly PheGlu Val Leu Phe Met Thr Glu Pro 485 490 495 Tyr Asp Glu Tyr Val Met GlnGln Val Lys Asp Phe Glu Asp Lys Lys 500 505 510 Phe Ala Cys Leu Thr LysGlu Gly Val His Phe Glu Glu Ser Glu Glu 515 520 525 Glu Lys Lys Gln ArgGlu Glu Glu Lys Ala Thr Cys Glu Lys Leu Cys 530 535 540 Lys Thr Met LysGlu Val Leu Gly Asp Lys Val Glu Lys Val Thr Val 545 550 555 560 Ser GluArg Leu Ser Thr Ser Pro Cys Ile Leu Val Thr Ser Glu Phe 565 570 575 GlyTrp Ser Ala His Met Glu Gln Met Met Arg Asn Gln Ala Leu Arg 580 585 590Asp Ser Ser Met Ala Gln Tyr Met Met Ser Lys Lys Thr Met Glu Leu 595 600605 Asn Pro Lys His Pro Ile Ile Lys Glu Leu Arg Arg Arg Val Glu Ala 610615 620 Asp Glu Asn Asp Lys Ala Val Lys Asp Leu Val Phe Leu Leu Phe Asp625 630 635 640 Thr Ser Leu Leu Thr Ser Gly Phe Gln Leu Glu Asp Pro ThrTyr Ala 645 650 655 Glu Arg Ile Asn Arg Met Ile Lys Leu Gly Leu Ser LeuAsp Glu Glu 660 665 670 Glu Glu Glu Glu Ala Val Glu Ala Ala Val Ala GluThr Ala Pro Ala 675 680 685 Glu Val Thr Ala Gly Thr Ser Ser Met Glu LeuVal Asp 690 695 700 <210> SEQ ID NO 17 <211> LENGTH: 704 <212> TYPE: PRT<213> ORGANISM: T. Cruzi <400> SEQUENCE: 17 Met Thr Glu Thr Phe Ala PheGln Ala Glu Ile Asn Gln Leu Met Ser 1 5 10 15 Leu Ile Ile Asn Thr PheTyr Ser Asn Lys Glu Ile Phe Leu Arg Glu 20 25 30 Leu Ile Ser Asn Ala SerAsp Ala Cys Asp Lys Ile Arg Tyr Gln Ser 35 40 45 Leu Thr Asn Gln Ala ValLeu Gly Asp Glu Ser His Leu Arg Ile Arg 50 55 60 Val Val Pro Asp Lys AlaAsn Lys Thr Leu Thr Val Glu Asp Thr Gly 65 70 75 80 Ile Gly Met Thr LysAla Glu Leu Val Asn Asn Leu Gly Thr Ile Ala 85 90 95 Arg Ser Gly Thr LysAla Phe Met Glu Ala Leu Glu Ala Gly Gly Asp 100 105 110 Met Ser Met IleGly Gln Phe Gly Val Gly Phe Tyr Ser Ala Tyr Leu 115 120 125 Val Ala AspArg Val Thr Val Val Ser Lys Asn Asn Asp Asp Glu Ala 130 135 140 Tyr ThrTrp Glu Ser Ser Ala Gly Gly Thr Phe Thr Val Thr Pro Thr 145 150 155 160Pro Asp Cys Asp Leu Lys Arg Gly Thr Arg Ile Val Leu His Leu Lys 165 170175 Glu Asp Gln Gln Glu Tyr Leu Glu Glu Arg Arg Leu Lys Asp Leu Ile 180185 190 Lys Lys His Ser Glu Phe Ile Gly Tyr Asp Ile Glu Leu Met Val Glu195 200 205 Lys Ala Thr Glu Lys Glu Val Thr Asp Glu Asp Glu Asp Glu AlaAla 210 215 220 Ala Thr Lys Asn Glu Glu Gly Glu Glu Pro Lys Val Glu GluVal Lys 225 230 235 240 Asp Asp Ala Glu Glu Gly Glu Lys Lys Lys Lys ThrLys Lys Val Lys 245 250 255 Glu Val Thr Gln Glu Phe Val Val Gln Asn LysHis Lys Pro Leu Trp 260 265 270 Thr Arg Asp Pro Lys Asp Val Thr Lys GluGlu Tyr Ala Ala Phe Tyr 275 280 285 Lys Ala Ile Ser Asn Asp Trp Glu GluPro Leu Ser Thr Lys His Phe 290 295 300 Ser Val Glu Gly Gln Leu Glu PheArg Ala Ile Leu Phe Val Pro Lys 305 310 315 320 Arg Ala Pro Phe Asp MetPhe Glu Pro Ser Lys Lys Arg Asn Asn Ile 325 330 335 Lys Leu Tyr Val ArgArg Val Phe Ile Met Asp Asn Cys Glu Asp Leu 340 345 350 Cys Pro Glu TrpLeu Ala Phe Val Arg Gly Val Val Asp Ser Glu Asp 355 360 365 Leu Pro LeuAsn Ile Ser Arg Glu Asn Leu Gln Gln Asn Lys Ile Leu 370 375 380 Lys ValIle Arg Lys Asn Ile Val Lys Lys Ala Leu Glu Leu Phe Glu 385 390 395 400Glu Ile Ala Glu Asn Lys Glu Asp Tyr Lys Lys Phe Tyr Glu Gln Phe 405 410415 Gly Lys Asn Val Lys Leu Gly Ile His Glu Asp Ser Ala Asn Arg Lys 420425 430 Lys Leu Met Glu Leu Leu Arg Phe His Ser Ser Glu Ser Gly Glu Asp435 440 445 Met Thr Thr Leu Lys Asp Tyr Val Thr Arg Met Lys Glu Gly GlnLys 450 455 460 Cys Ile Tyr Tyr Val Thr Gly Asp Ser Lys Lys Lys Leu GluThr Ser 465 470 475 480 Pro Phe Ile Glu Gln Ala Arg Arg Arg Gly Phe GluVal Leu Phe Met 485 490 495 Thr Glu Pro Ile Asp Glu Tyr Val Met Gln GlnVal Lys Asp Phe Glu 500 505 510 Asp Lys Lys Phe Ala Cys Leu Thr Lys GluGly Val His Phe Glu Glu 515 520 525 Thr Glu Glu Glu Lys Lys Gln Arg GluGlu Glu Lys Thr Ala Tyr Glu 530 535 540 Arg Leu Cys Lys Ala Met Lys AspVal Leu Gly Asp Lys Val Glu Lys 545 550 555 560 Val Val Val Ser Glu ArgLeu Ala Thr Ser Pro Cys Ile Leu Val Thr 565 570 575 Ser Glu Phe Gly TrpSer Ala His Met Glu Gln Ile Met Arg Asn Gln 580 585 590 Ala Leu Arg AspSer Ser Met Ser Ala Tyr Met Met Ser Lys Lys Thr 595 600 605 Met Glu IleAsn Pro Ala His Pro Ile Val Lys Glu Leu Lys Arg Arg 610 615 620 Val GluAla Asp Glu Asn Asp Lys Ala Val Lys Asp Leu Val Tyr Leu 625 630 635 640Leu Phe Asp Thr Ala Leu Leu Thr Ser Gly Phe Thr Leu Asp Asp Pro 645 650655 Thr Ser Tyr Ala Glu Arg Ile His Arg Met Ile Lys Leu Gly Leu Ser 660665 670 Leu Asp Asp Glu Asp Asn Gly Asn Glu Glu Ala Glu Pro Ala Ala Ala675 680 685 Val Pro Ala Glu Pro Val Ala Gly Thr Ser Ser Met Glu Gln ValAsp 690 695 700 <210> SEQ ID NO 18 <211> LENGTH: 732 <212> TYPE: PRT<213> ORGANISM: Homo sapien <400> SEQUENCE: 18 Met Pro Glu Glu Thr GlnThr Gln Asp Gln Pro Met Glu Glu Glu Glu 1 5 10 15 Val Glu Thr Phe AlaPhe Gln Ala Glu Ile Ala Gln Leu Met Ser Leu 20 25 30 Ile Ile Asn Thr PheTyr Ser Asn Lys Glu Ile Phe Leu Arg Glu Leu 35 40 45 Ile Ser Asn Ser SerAsp Ala Leu Asp Lys Ile Arg Tyr Glu Ser Leu 50 55 60 Thr Asp Pro Ser LysLeu Asp Ser Gly Lys Glu Leu His Ile Asn Leu 65 70 75 80 Ile Pro Asn LysGln Asp Arg Ala Leu Thr Ile Val Asp Thr Gly Ile 85 90 95 Gly Met Thr LysAla Asp Leu Ile Asn Asn Leu Gly Thr Ile Ala Lys 100 105 110 Ser Gly ThrLys Ala Phe Met Glu Ala Leu Gln Ala Gly Ala Asp Ile 115 120 125 Ser MetIle Gly Gln Phe Gly Val Gly Phe Tyr Ser Ala Tyr Leu Val 130 135 140 AlaGlu Lys Val Thr Val Ile Thr Lys His Asn Asp Asp Glu Gln Tyr 145 150 155160 Ala Trp Glu Ser Ser Ala Gly Gly Ser Phe Thr Val Arg Thr Asp Thr 165170 175 Gly Glu Pro Met Gly Arg Gly Thr Lys Val Ile Leu His Leu Lys Glu180 185 190 Asp Gln Thr Glu Tyr Leu Glu Glu Arg Arg Ile Lys Glu Ile ValLys 195 200 205 Lys His Ser Gln Phe Ile Gly Tyr Pro Ile Thr Leu Phe ValGlu Lys 210 215 220 Glu Arg Asp Lys Glu Val Ser Asp Asp Glu Ala Glu GluLys Glu Asp 225 230 235 240 Lys Glu Glu Glu Lys Glu Lys Glu Glu Lys GluSer Glu Asp Lys Pro 245 250 255 Glu Ile Glu Asp Val Gly Ser Asp Glu GluAsp Glu Lys Lys Asp Gly 260 265 270 Asp Lys Lys Lys Lys Lys Lys Ile LysGlu Lys Tyr Ile Asp Lys Glu 275 280 285 Glu Leu Asn Lys Thr Lys Pro IleTrp Thr Arg Asn Pro Asp Asp Ile 290 295 300 Thr Asn Glu Glu Tyr Gly GluPhe Tyr Lys Ser Leu Thr Asn Asp Trp 305 310 315 320 Glu Asp His Leu AlaVal Lys His Phe Ser Val Glu Gly Gln Leu Glu 325 330 335 Phe Arg Ala LeuLeu Phe Val Pro Arg Arg Ala Pro Phe Asp Leu Phe 340 345 350 Glu Asn ArgLys Lys Lys Asn Asn Ile Lys Leu Tyr Val Arg Arg Val 355 360 365 Phe IleMet Asp Asn Cys Glu Glu Leu Ile Pro Glu Tyr Leu Asn Phe 370 375 380 IleArg Gly Val Val Asp Ser Glu Asp Leu Pro Leu Asn Ile Ser Arg 385 390 395400 Glu Met Leu Gln Gln Ser Lys Ile Leu Lys Val Ile Arg Lys Asn Leu 405410 415 Val Lys Lys Cys Leu Glu Leu Phe Thr Glu Leu Ala Glu Asp Lys Glu420 425 430 Asn Tyr Lys Lys Phe Tyr Glu Gln Phe Ser Lys Asn Ile Lys LeuGly 435 440 445 Ile His Glu Asp Ser Gln Asn Arg Lys Lys Leu Ser Glu LeuLeu Arg 450 455 460 Tyr Tyr Thr Ser Ala Ser Gly Asp Glu Met Val Ser LeuLys Asp Tyr 465 470 475 480 Cys Thr Arg Met Lys Glu Asn Gln Lys His IleTyr Tyr Ile Thr Gly 485 490 495 Glu Thr Lys Asp Gln Val Ala Asn Ser AlaPhe Val Glu Arg Leu Arg 500 505 510 Lys His Gly Leu Glu Val Ile Tyr MetIle Glu Pro Ile Asp Glu Tyr 515 520 525 Cys Val Gln Gln Leu Lys Glu PheGlu Gly Lys Thr Leu Val Ser Val 530 535 540 Thr Lys Glu Gly Leu Glu LeuPro Glu Asp Glu Glu Glu Lys Lys Lys 545 550 555 560 Gln Glu Glu Lys LysThr Lys Phe Glu Asn Leu Cys Lys Ile Met Lys 565 570 575 Asp Ile Leu GluLys Lys Val Glu Lys Val Val Val Ser Asn Arg Leu 580 585 590 Val Thr SerPro Cys Cys Leu Val Thr Ser Thr Tyr Gly Trp Thr Ala 595 600 605 Asn MetGlu Arg Ile Met Lys Ala Gln Ala Leu Arg Asp Asn Ser Thr 610 615 620 MetGly Tyr Met Ala Ala Lys Lys His Leu Glu Ile Asn Pro Asp His 625 630 635640 Ser Ile Ile Glu Thr Leu Arg Gln Lys Ala Glu Ala Asp Lys Asn Asp 645650 655 Lys Ser Val Lys Asp Leu Val Ile Leu Leu Tyr Glu Thr Ala Leu Leu660 665 670 Ser Ser Gly Phe Ser Leu Glu Asp Pro Gln Thr His Ala Asn ArgIle 675 680 685 Tyr Arg Met Ile Lys Leu Gly Leu Gly Ile Asp Glu Asp AspPro Thr 690 695 700 Ala Asp Asp Thr Ser Ala Ala Val Thr Glu Glu Met ProPro Leu Glu 705 710 715 720 Gly Asp Asp Asp Thr Ser Arg Met Glu Glu ValAsp 725 730 <210> SEQ ID NO 19 <211> LENGTH: 1019 <212> TYPE: DNA <213>ORGANISM: Leishmania major <400> SEQUENCE: 19 gaattcggca cgaggtttctgtactttatt gcttccagcc tttattcact cttcgatttc 60 ctctaacacc atgtcctccgagcgcacctt tattgccgtc aagccggacg gcgtgcagcg 120 cggcctcgtt ggcgagatcatcgcccgctt cgagcgcaag ggctacaagc tcgtcgcctt 180 gaagatactg cagccgacgacggagcaggc ccagggtcac tataaggacc tttgctccaa 240 gccgtttttc ccggcccttgtgaagtactt ctcctctggc ccgatcgtgt gtatggtgtg 300 ggagggtaag aacgtggtgaagagcggccg cgtgctgctc ggcgcgacga acccggccga 360 ctcacagccc ggcacgatccgtggcgactt tgccgtggat gtgggccgca acgtgtgcca 420 cgggtccgac tctgtggagagcgcggagcg cgagatcgcc ttttggttca aggcggatga 480 gatcgcgagc tggacgtcgcactccgtgtc ccagatctat gagtaacggt gattgcggac 540 acgctttgag gacgtagctgtacccccaat gaattcttct ctgaaaacca catcataagc 600 ctcttaagag gttatttttcttgatcgatg cccggtggtg accagcacca ttcctttatc 660 ggattcactc acactcctagcgaatcatgt agtgcggtga gagtgggctc tggaggagac 720 tgttgtgtag ccatggcttcaggagagaaa acaaaataca aggaaaggca atatgtaact 780 atggggttcc cttttttactatgcaaagtt tttataactc ctgatcggca aaaacaacaa 840 caaccgccat acaccaagagcaaatgcttt cttctgcgga ctgtgcttct gttttttttt 900 atgaaggagt gactcgcgcgatgaaaagtg tgtgcgtggg agatgtattt cctttttttg 960 ttcatagtgg cgacagctcactgttgacga tgacaaaaaa aaaaaaaaaa aaactcgag 1019 <210> SEQ ID NO 20 <211>LENGTH: 151 <212> TYPE: PRT <213> ORGANISM: Leishmania major <400>SEQUENCE: 20 Met Ser Ser Glu Arg Thr Phe Ile Ala Val Lys Pro Asp Gly ValGln 1 5 10 15 Arg Gly Leu Val Gly Glu Ile Ile Ala Arg Phe Glu Arg LysGly Tyr 20 25 30 Lys Leu Val Ala Leu Lys Ile Leu Gln Pro Thr Thr Glu GlnAla Gln 35 40 45 Gly His Tyr Lys Asp Leu Cys Ser Lys Pro Phe Phe Pro AlaLeu Val 50 55 60 Lys Tyr Phe Ser Ser Gly Pro Ile Val Cys Met Val Trp GluGly Lys 65 70 75 80 Asn Val Val Lys Ser Gly Arg Val Leu Leu Gly Ala ThrAsn Pro Ala 85 90 95 Asp Ser Gln Pro Gly Thr Ile Arg Gly Asp Phe Ala ValAsp Val Gly 100 105 110 Arg Asn Val Cys His Gly Ser Asp Ser Val Glu SerAla Glu Arg Glu 115 120 125 Ile Ala Phe Trp Phe Lys Ala Asp Glu Ile AlaSer Trp Thr Ser His 130 135 140 Ser Val Ser Gln Ile Tyr Glu 145 150<210> SEQ ID NO 21 <211> LENGTH: 1523 <212> TYPE: DNA <213> ORGANISM:Leishmania major <400> SEQUENCE: 21 gaattcggca cgagtgctgc ccgacatgacatgctcgctg accggacttc agtgcacaga 60 cccgaactgc aagacctgca caacttacggtcagtgcaca gactgcaacg acggctacgg 120 tctcacctcc tccagcgttt gcgtgcgctgcagtgtagcg ggctgcaaga gctgccccgt 180 cgacgctaac gtctgcaaag tgtgtctcggcggcagcgag ccgatcaaca atatgtgccc 240 ctgcaccgac cccaactgcg ccagctgccccagcgacgct ggcacgtgca ctcagtgcgc 300 gaacggctac ggtctcgtgg acggcgcctgtgtgagatgc caggagccca actgcttcag 360 ctgcgacagc gacgcgaata agtgcacacaatgtgcgccg aactactacc tcaccccgct 420 cttgacctgc tccccggtgg cctgcaacatcgagcactgc atgcagtgcg acccacagac 480 gccgtcgcgc tgccaggagt gcgtgtccccctacgtggtt gacagctacg acggcctctg 540 caggctctcc gatgcctgct ccgtgcccaactgcaagaag tgcgagaccg gtacctccag 600 gctctgcgcc gagtgcgaca ccggctacagtctctccgcc gacgcgacga gctgcagcag 660 tccaaccacg cagccgtgcg aggtggagcactgcaacaca tgtgtgaacg gcgatagcac 720 ccgctgtgcc tactgcaaca ccggctactacgtctccgat ggcaagtgca aggccatgca 780 gggctgctac gtgtcgaact gcgcgcagtgcatgctgctt gacagcacca agtgctccac 840 gtgcgtgaaa gggtacctgc tcacgtcgtcctacagttgc gtctcgcaga aagtcatcaa 900 cagtgcggcc gcgccctact ctctgtgggtggccgccgcc gtgctcctca cctcttttgc 960 catgcaccta gcatagtgcg cagcggcatgcgaacaaccc cactctcatt ctccaacatg 1020 tgcatacaca cacacacaga cagcggggcagcaccccctc cccacacaca cacacgcact 1080 tcccccttgt cttgttcttc tttcctcgttcgcatttctt tctctcgtgc gctggcgccg 1140 gcctcctgca cgtcgctccc ctccccctaacctctattct ctctctctct ctctctcgcc 1200 ggcatcattg cttcttaccc ttttctgatccttgctcgcg tgggcggaca ctgccacagt 1260 cccacagcgc agacacacgt gtttaaacggcgcaggcatc cctccctatc acttcatttc 1320 tcctaaagcc actcaccaag tcgcacaccgccctccccca tcggccgccc ttccgggcgc 1380 agctgtgcgg aatgggtgtg tgctcgacctcgttcctggc agctcactcg catgtgtaca 1440 gccactccaa ccacgaaagc tctcttctgcgcacataaaa aaaaaaaaaa aaaaaaaact 1500 cgaggggggg cccggtaccc aaa 1523<210> SEQ ID NO 22 <211> LENGTH: 320 <212> TYPE: PRT <213> ORGANISM:Leishmania major <400> SEQUENCE: 22 Val Leu Pro Asp Met Thr Cys Ser LeuThr Gly Leu Gln Cys Thr Asp 1 5 10 15 Pro Asn Cys Lys Thr Cys Thr ThrTyr Gly Gln Cys Thr Asp Cys Asn 20 25 30 Asp Gly Tyr Gly Leu Thr Ser SerSer Val Cys Val Arg Cys Ser Val 35 40 45 Ala Gly Cys Lys Ser Cys Pro ValAsp Ala Asn Val Cys Lys Val Cys 50 55 60 Leu Gly Gly Ser Glu Pro Ile AsnAsn Met Cys Pro Cys Thr Asp Pro 65 70 75 80 Asn Cys Ala Ser Cys Pro SerAsp Ala Gly Thr Cys Thr Gln Cys Ala 85 90 95 Asn Gly Tyr Gly Leu Val AspGly Ala Cys Val Arg Cys Gln Glu Pro 100 105 110 Asn Cys Phe Ser Cys AspSer Asp Ala Asn Lys Cys Thr Gln Cys Ala 115 120 125 Pro Asn Tyr Tyr LeuThr Pro Leu Leu Thr Cys Ser Pro Val Ala Cys 130 135 140 Asn Ile Glu HisCys Met Gln Cys Asp Pro Gln Thr Pro Ser Arg Cys 145 150 155 160 Gln GluCys Val Ser Pro Tyr Val Val Asp Ser Tyr Asp Gly Leu Cys 165 170 175 ArgLeu Ser Asp Ala Cys Ser Val Pro Asn Cys Lys Lys Cys Glu Thr 180 185 190Gly Thr Ser Arg Leu Cys Ala Glu Cys Asp Thr Gly Tyr Ser Leu Ser 195 200205 Ala Asp Ala Thr Ser Cys Ser Ser Pro Thr Thr Gln Pro Cys Glu Val 210215 220 Glu His Cys Asn Thr Cys Val Asn Gly Asp Ser Thr Arg Cys Ala Tyr225 230 235 240 Cys Asn Thr Gly Tyr Tyr Val Ser Asp Gly Lys Cys Lys AlaMet Gln 245 250 255 Gly Cys Tyr Val Ser Asn Cys Ala Gln Cys Met Leu LeuAsp Ser Thr 260 265 270 Lys Cys Ser Thr Cys Val Lys Gly Tyr Leu Leu ThrSer Ser Tyr Ser 275 280 285 Cys Val Ser Gln Lys Val Ile Asn Ser Ala AlaAla Pro Tyr Ser Leu 290 295 300 Trp Val Ala Ala Ala Val Leu Leu Thr SerPhe Ala Met His Leu Ala 305 310 315 320 <210> SEQ ID NO 23 <211> LENGTH:797 <212> TYPE: DNA <213> ORGANISM: Leishmania major <400> SEQUENCE: 23ctgtacttta ttgccaccag ccagccatgt cctgcggtaa cgccaagatc aactctcccg 60cgccgtcctt cgaggaggtg gcgctcatgc ccaacggcag cttcaagaag atcagcctct 120cctcctacaa gggcaagtgg gtcgtgctct tcttctaccc gctcgacttt agcttcgtgt 180gcccgacaga ggtcatcgcg ttctccgaca gcgtgagtcg cttcaacgag ctcaactgcg 240aggtcctcgc gtgctcgata gacagcgagt acgcgcacct gcagtggacg ctgcaggacc 300gcaagaaggg cggcctcggg accatggcga tcccaatgct agccgacaag accaagagca 360tcgctcgttc ctacggcgtg ctggaggaga gccagggcgt ggcctaccgc ggtctcttca 420tcatcgaccc ccatggcatg ctgcgtcaga tcaccgtcaa tgacatgccg gtgggccgca 480gcgtggagga ggttctacgc ctgctggagg cttttcagtt cgtggagaag cacggcgagg 540tgtgccccgc gaactggaag aagggcgccc ccacgatgaa gccggaaccg aatgcgtctg 600tcgagggata cttcagcaag cagtaaacct gtgagcgtcg caggagtcag tgtgacctca 660cccgcctctg ccagtgggtg cgagagggcg tgagggattg tgggaaggct gttggatatg 720atgcagacag cgatgaatgc aactcccaca cactggccct cctcagccct ctccacacag 780acacacgcac gcatgtg 797 <210> SEQ ID NO 24 <211> LENGTH: 199 <212> TYPE:PRT <213> ORGANISM: Leishmania major <400> SEQUENCE: 24 Met Ser Cys GlyAsn Ala Lys Ile Asn Ser Pro Ala Pro Ser Phe Glu 1 5 10 15 Glu Val AlaLeu Met Pro Asn Gly Ser Phe Lys Lys Ile Ser Leu Ser 20 25 30 Ser Tyr LysGly Lys Trp Val Val Leu Phe Phe Tyr Pro Leu Asp Phe 35 40 45 Ser Phe ValCys Pro Thr Glu Val Ile Ala Phe Ser Asp Ser Val Ser 50 55 60 Arg Phe AsnGlu Leu Asn Cys Glu Val Leu Ala Cys Ser Ile Asp Ser 65 70 75 80 Glu TyrAla His Leu Gln Trp Thr Leu Gln Asp Arg Lys Lys Gly Gly 85 90 95 Leu GlyThr Met Ala Ile Pro Met Leu Ala Asp Lys Thr Lys Ser Ile 100 105 110 AlaArg Ser Tyr Gly Val Leu Glu Glu Ser Gln Gly Val Ala Tyr Arg 115 120 125Gly Leu Phe Ile Ile Asp Pro His Gly Met Leu Arg Gln Ile Thr Val 130 135140 Asn Asp Met Pro Val Gly Arg Ser Val Glu Glu Val Leu Arg Leu Leu 145150 155 160 Glu Ala Phe Gln Phe Val Glu Lys His Gly Glu Val Cys Pro AlaAsn 165 170 175 Trp Lys Lys Gly Ala Pro Thr Met Lys Pro Glu Pro Asn AlaSer Val 180 185 190 Glu Gly Tyr Phe Ser Lys Gln 195 <210> SEQ ID NO 25<211> LENGTH: 637 <212> TYPE: DNA <213> ORGANISM: Leishmania tropica<400> SEQUENCE: 25 ttacatatgc atcaccacca ccaccacatg tcctgcggtaacgccaagat caactctccc 60 gcgccgccct tcgaggagat ggcgctcatg cccaacggcagcttcaagaa gatcagcctc 120 tccgcctaca agggcaagtg ggtcgtgctc ttcttctacccgctcgactt caccttcgtg 180 tgcccgacag agatcatcgc gttctccgac aacgtgagtcgcttcaacga gctcaactgc 240 gaggtcctcg cgtgctcgat ggacagcgag tacgcgcacctgcagtggac gctgcaggac 300 cgcaagaagg gcggcctcgg ggccatggcg atcccaatgctggccgacaa gactaagagc 360 atcgctcgtt cctacggcgt gctggaggag agccagggcgtggcctaccg cggtctcttc 420 atcatcgacc cccgtggcat ggtgcgtcag atcaccgtcaacgacatgcc ggtgggccgc 480 aacgtggagg aggctctgcg cctgctggag gctttgcagttcgtggagaa gcacggcgag 540 gtgtgccccg cgaactggaa gaagggcgcc cccacgatgaagccggaacc gaaggcgtct 600 gtcgagggat acttcagcaa gcagtaagaa ttccatg 637<210> SEQ ID NO 26 <211> LENGTH: 206 <212> TYPE: PRT <213> ORGANISM:Leishmania tropica <400> SEQUENCE: 26 Met His His His His His His MetSer Cys Gly Asn Ala Lys Ile Asn 1 5 10 15 Ser Pro Ala Pro Pro Phe GluGlu Met Ala Leu Met Pro Asn Gly Ser 20 25 30 Phe Lys Lys Ile Ser Leu SerAla Tyr Lys Gly Lys Trp Val Val Leu 35 40 45 Phe Phe Tyr Pro Leu Asp PheThr Phe Val Cys Pro Thr Glu Ile Ile 50 55 60 Ala Phe Ser Asp Asn Val SerArg Phe Asn Glu Leu Asn Cys Glu Val 65 70 75 80 Leu Ala Cys Ser Met AspSer Glu Tyr Ala His Leu Gln Trp Thr Leu 85 90 95 Gln Asp Arg Lys Lys GlyGly Leu Gly Ala Met Ala Ile Pro Met Leu 100 105 110 Ala Asp Lys Thr LysSer Ile Ala Arg Ser Tyr Gly Val Leu Glu Glu 115 120 125 Ser Gln Gly ValAla Tyr Arg Gly Leu Phe Ile Ile Asp Pro Arg Gly 130 135 140 Met Val ArgGln Ile Thr Val Asn Asp Met Pro Val Gly Arg Asn Val 145 150 155 160 GluGlu Ala Leu Arg Leu Leu Glu Ala Leu Gln Phe Val Glu Lys His 165 170 175Gly Glu Val Cys Pro Ala Asn Trp Lys Lys Gly Ala Pro Thr Met Lys 180 185190 Pro Glu Pro Lys Ala Ser Val Glu Gly Tyr Phe Ser Lys Gln 195 200 205<210> SEQ ID NO 27 <211> LENGTH: 51 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR primer<400> SEQUENCE: 27 caattacata tgcatcacca tcaccatcac atgtcctgcggtaacgccaa g 51 <210> SEQ ID NO 28 <211> LENGTH: 31 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: PCR primer <400> SEQUENCE: 28 catggaattc ttactgcttgctgaagtatc c 31 <210> SEQ ID NO 29 <211> LENGTH: 520 <212> TYPE: DNA<213> ORGANISM: Leishmania major <220> FEATURE: <221> NAME/KEY:misc_feature <222> LOCATION: (1)...(520) <223> OTHER INFORMATION: n = A,T, C or G <400> SEQUENCE: 29 ggcacgagcc cttgcctaca tttgctcgcc gatattcgcggggagttctt caatttgcgt 60 cgcgtagaac tgctcaatgt cgcgcaacaa gcgcagctcgtcgtggcgca cgaaggtgat 120 ggccagtcca gtgcggccca tgcggccagt gcggccgatgcggtgaatgt actgctcacg 180 cgcgagcggc aaatcgtagc tgaggacgag cgagacgcgctccacatcaa tgccacgcgc 240 ccacaggtcc gttgtaatga ncacgcggct gtgtccattacggaatgccg cataatctcg 300 tcgcgctccg cctggggcat gtcgccgtgc atggcggacacagcgaaatt ctcgcgcgtc 360 atcttcttgg caagctgctc cacctttttg cgggtgttgcanaaaaccac ngcgtgggcg 420 atcgttaagc tgtcgtacaa actccatcaa gaaatcgaatttgtttttct cttcgtcnac 480 nganacaaan tactgtttaa cgctntccac ggtgatctca520 <210> SEQ ID NO 30 <211> LENGTH: 600 <212> TYPE: DNA <213> ORGANISM:Leishmania major <220> FEATURE: <221> NAME/KEY: misc_feature <222>LOCATION: (1)...(600) <223> OTHER INFORMATION: n = A, T, C or G <400>SEQUENCE: 30 ggcacaaggt tttcgggtta tcttcacgca tggtggagcg cagatgggtgaagtaaatac 60 gcggaccgaa ctgcttgatc atatcaacca gatcgttgtc agcacgcacgccgtangaac 120 cggtgcacat ggtaaaaccg tntgccatgc tgtttacggt atcaaccatccactgcatat 180 cttcaatggt ggaaacaatg cgcggcaggc cgaggatccg gcgcggctcatcatnnagnt 240 natnaaccan tcgcacgtct anttctgcac taaactacaa ntatcggtnacatatnataa 300 ggccnatttt cggtccagga ntatgtnctn tcaaaatgcc ncgttanncactcttaaatg 360 tctcangngn aaantngttc taaagggtgt ccaaaanntn nttaccnttccccncttact 420 tcaananctc ctcnaattcc cnggcccttn gacnannatt tnctattaaaanatanaann 480 ttcaaattna ttcccnacct nccntnncca aanntancna ataatcanncccctntcann 540 anntcccanc ttaccctccn ntngnngggn nnnccnattn ccccaancccncnctaaata 600 210> SEQ ID NO 31 <211> LENGTH: 600 <212> TYPE: DNA <213>ORGANISM: Leishmania major <220> FEATURE: <221> NAME/KEY: misc_feature<222> LOCATION: (1)...(600) <223> OTHER INFORMATION: n = A, T, C or G<400> SEQUENCE: 31 ggcacgagcc tcagtggagc tcaatgaaga tattgcagtatcttactctg gatggcactc 60 aggtctccgg cacgctgccg ccccagtgga gcgcgatggcatcggtgcga attcttaacc 120 tgnagggtac tgaggtctct ggtacgctgc cgcctgagtggatatcnatg ancaggctgc 180 aaactctgaa tctgcggcgc acgaaantat ccggcactctgccgcccgaa tgganttcta 240 tgaacagcct ggagtacttt cacctttatc ttactcaggtctccggcacg ctgccgcccg 300 agtggagtgg gatgtcnaag gccgcatact tctggctggaatactgcgac ctgtccggca 360 ntctgccgcc cnagtggtcg tcnatgccaa agctgcgcggtatctcactg ancggcaaca 420 aattcttgcg ngtgtntncc ngactcntgg gattcagaaaggtggtcctt gttgttgggc 480 atcnaaggan caaaccccaa ngggcccncn aattgcttgggcntgcttaa gganttgcac 540 naaccaacnc cnccaaaaac cccccccacc ncnaaannacnancccccac ttaanncccn 600 <210> SEQ ID NO 32 <211> LENGTH: 600 <212>TYPE: DNA <213> ORGANISM: Leishmania major <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (1)...(600) <223> OTHERINFORMATION: n = A,T,C or G <400> SEQUENCE: 32 ngcacgagaa gcgcaactggcgcatcgcat ctgtgactat ctgcctgaac aggggcaatn 60 gtttgttggt aacagcctggtggtacgtct gattgatncg cttncgcaan ttccggcagg 120 ttacccggtg tacancaaccgtggggccan cggtatcnac nggctgcttt cgaccgccgc 180 cggngttcan cgggcaancggcaaaccgac gctggcgatt gtgggcgatc tctccgcact 240 ttacgatctc aacgcnctggcgttattgcg tcaggtttct gcgccgctgg tattaattgt 300 ggtgaacaac aacggcngggcaaaattttc tcgctgttgc caacgccccc aaagcnagcg 360 tgaagcgttt ctatctgatgccgcaaaacg tccattttga aacacgccgc cncccatgtt 420 tcganctgaa aatatcatcgtccgcaaaac tggcangaaa cttngaaaac cgcattttgc 480 cgacnccctg gcncacgcccaacccaccca ccggttgatt gaaaatggtg ggttaacgaa 540 nccnnatggg tgccccaaancncnnccanc caaatttctg ggcccaggtt aaancccttt 600 <210> SEQ ID NO 33 <211>LENGTH: 600 <212> TYPE: DNA <213> ORGANISM: Leishmania major <220>FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (1)...(600) <223>OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 33 acgatgaccatgccccgaag gaggatggcc atgcgccgaa gaacgatgac catgccccga 60 aggaggatggccatgcgccg aagaacgatg accatgcccc gaaggaggat ggccatgcgc 120 cgaagaacgacggggatgtg cagaanaaga gcgaagatgg agacaacgtg ggagagggag 180 gcaagggcaatgaggatggt aacgatgatc agccgaagga gcacgctgcc ggcaactagt 240 gggctgcgtccgggcttgtg tgcganccgt gctctgcacc ccgccgctcg tgcatcctcg 300 catgtggactgcgtgtgtct ctcccgcttt gtctctctcc cccacacagt ggctgatgcc 360 tgcacggggttgctgtggct gcacctcctg accactgcca gctttcttgg cttgcctccc 420 ctctgcgcctccgctcgtgc cgctcgtgcc gaattcgata tcaagcttat cgataccgtc 480 nacctcgaaggggggcccgg ttacccattc gccctatant gagtcntatt acaattcctg 540 gcgtcgttttacacgtcgtg actgggaaaa accctggcgt tccccactta tcgccttgca 600 <210> SEQ IDNO 34 <211> LENGTH: 516 <212> TYPE: DNA <213> ORGANISM: Leishmania major<400> SEQUENCE: 34 agctgcagca gcgcctagac accgccacgc agcagcgcgccgagctggag gcacgggtgg 60 cacggctggc cgcggaccgc gacgaggcgc gccagcagctggccgcgaac gccgaggagc 120 tgcagcagcg cctagacacc gccacgcagc agcgcgccgagctggaggca cgggtggcac 180 ggctggccgc ggacggcgac gaggcccgcc agcagctggccgcgaacgcc gaggagctgc 240 agcagcgcct agacaccgcc acgcagcagc gcgccgagctggaggcacag gtggcacggc 300 tggccgcgaa cgccgaggag ctgcagcagc gcctagacaccgccacgcag cagcgcgccg 360 agctggaggc acgggtggca cggctggccg cggaccgcgacgaggcgcgc cagcagctgg 420 ccgcgaacgc cgaggagctg cagcagcgcc tagacaccgccacgcagcag cgcgccgagc 480 tggargcaca ggtggcacgg ctggccgcga amgccg 516<210> SEQ ID NO 35 <211> LENGTH: 822 <212> TYPE: DNA <213> ORGANISM:Leishmania major <220> FEATURE: <221> NAME/KEY: misc_feature <222>LOCATION: (1)...(822) <223> OTHER INFORMATION: n = A,T,C or G <400>SEQUENCE: 35 ggcacganag atcttcgtga agacgctgac cggcaanacg atcgcgctggaggtggagcc 60 gagcgacacg atcgagaacg tgaaggccaa gatccaggac aaggagggcatcccgccgga 120 ccagcagcgc ctgatcttcg ccggcaagca gctggaggan ggccgcacgctctcggacta 180 caacatccag aaggagtcca cgctgcacct ggtgctgcgc ctgcgcggcggcatgcanat 240 cttcgtgaaa acgctnaccg gcaanacaat cgcgctggaa gtggagccgaacgaccnatc 300 gaaaacgtga aggccnanat ccangacaag gaaggcntcc cgccggancagcacgcctga 360 tcttccnccg gcaaccactt gangaagggc ncacgctctc ngactacnacatccanaaag 420 gattccnccc tgcaccttgt tgcttgcncc ttgctcgggg ggcatgccnaatcttccttn 480 aaaacctcaa ccggcaanaa caatcccccn cngaagttgg aacccaaccancccattcna 540 aaactttaaa ggccnnnatt ccngaacaan gaagggcttc ccccccggaccnncaancnc 600 cctgattntt cccccggnnn ncantttgga angaagggcc ccnccctccnccgaattncn 660 acntcccnaa anggattccc cccctnccct tgntttttgc gccnnnnnncggcnncntnc 720 cnaaattccg nccnaaggnc cccantanan cnactttccc nttcccccccnnnnttttgc 780 ntaaantttt tncccccnna aanntcccnt ttncnanttn an 822 <210>SEQ ID NO 36 <211> LENGTH: 146 <212> TYPE: PRT <213> ORGANISM:Leishmania major <220> FEATURE: <221> NAME/KEY: VARIANT <222> LOCATION:(1)...(146) <223> OTHER INFORMATION: Xaa = Any Amino Acid <400>SEQUENCE: 36 Gly Thr Ser Pro Cys Leu His Leu Leu Ala Asp Ile Arg Gly GluPhe 1 5 10 15 Phe Asn Leu Arg Arg Val Glu Leu Leu Asn Val Ala Gln GlnAla Gln 20 25 30 Leu Val Val Ala His Glu Gly Asp Gly Gln Ser Ser Ala AlaHis Ala 35 40 45 Ala Ser Ala Ala Asp Ala Val Asn Val Leu Leu Thr Arg GluArg Gln 50 55 60 Ile Val Ala Glu Asp Glu Arg Asp Ala Leu His Ile Asn AlaThr Arg 65 70 75 80 Pro Gln Val Arg Cys Asn Xaa His Ala Ala Val Ser IleThr Glu Cys 85 90 95 Arg Ile Ile Ser Ser Arg Ser Ala Trp Gly Met Ser ProCys Met Ala 100 105 110 Asp Thr Ala Lys Phe Ser Arg Val Ile Phe Leu AlaSer Cys Ser Thr 115 120 125 Phe Leu Arg Val Leu Xaa Lys Thr Thr Ala TrpAla Ile Val Lys Leu 130 135 140 Ser Tyr 145 <210> SEQ ID NO 37 <211>LENGTH: 77 <212> TYPE: PRT <213> ORGANISM: Leishmania major <220>FEATURE: <221> NAME/KEY: VARIANT <222> LOCATION: (1)...(77) <223> OTHERINFORMATION: Xaa = Any Amino Acid <400> SEQUENCE: 37 Ala Gln Gly Phe ArgVal Ile Phe Thr His Gly Gly Ala Gln Met Gly 1 5 10 15 Glu Val Asn ThrArg Thr Glu Leu Leu Asp His Ile Asn Gln Ile Val 20 25 30 Val Ser Thr HisAla Val Xaa Thr Gly Ala His Gly Lys Thr Val Cys 35 40 45 His Ala Val TyrGly Ile Asn His Pro Leu His Ile Phe Asn Gly Gly 50 55 60 Asn Asn Ala ArgGln Ala Glu Asp Pro Ala Arg Leu Ile 65 70 75 <210> SEQ ID NO 38 <211>LENGTH: 68 <212> TYPE: PRT <213> ORGANISM: Leishmania major <220>FEATURE: <221> NAME/KEY: VARIANT <222> LOCATION: (1)...(68) <223> OTHERINFORMATION: Xaa = Any Amino Acid <400> SEQUENCE: 38 His Glu Pro Gln TrpSer Ser Met Lys Ile Leu Gln Tyr Leu Thr Leu 1 5 10 15 Asp Gly Thr GlnVal Ser Gly Thr Leu Pro Pro Gln Trp Ser Ala Met 20 25 30 Ala Ser Val ArgIle Leu Asn Leu Xaa Gly Thr Glu Val Ser Gly Thr 35 40 45 Leu Pro Pro GluTrp Ile Ser Met Xaa Arg Leu Gln Thr Leu Asn Leu 50 55 60 Arg Arg Thr Lys65 <210> SEQ ID NO 39 <211> LENGTH: 65 <212> TYPE: PRT <213> ORGANISM:Leishmania major <220> FEATURE: <221> NAME/KEY: VARIANT <222> LOCATION:(1)...(65) <223> OTHER INFORMATION: Xaa = Any Amino Acid <400> SEQUENCE:39 Ala Arg Glu Ala Gln Leu Ala His Arg Ile Cys Asp Tyr Leu Pro Glu 1 510 15 Gln Gly Gln Xaa Phe Val Gly Asn Ser Leu Val Val Arg Leu Ile Asp 2025 30 Xaa Leu Xaa Gln Xaa Pro Ala Gly Tyr Pro Val Tyr Xaa Asn Arg Gly 3540 45 Ala Xaa Gly Ile Xaa Xaa Leu Leu Ser Thr Ala Ala Gly Val Xaa Arg 5055 60 Ala 65 <210> SEQ ID NO 40 <211> LENGTH: 78 <212> TYPE: PRT <213>ORGANISM: Leishmania major <220> FEATURE: <221> NAME/KEY: VARIANT <222>LOCATION: (1)...(78) <223> OTHER INFORMATION: Xaa = Any Amino Acid <400>SEQUENCE: 40 Asp Asp His Ala Pro Lys Glu Asp Gly His Ala Pro Lys Asn AspAsp 1 5 10 15 His Ala Pro Lys Glu Asp Gly His Ala Pro Lys Asn Asp AspHis Ala 20 25 30 Pro Lys Glu Asp Gly His Ala Pro Lys Asn Asp Gly Asp ValGln Xaa 35 40 45 Lys Ser Glu Asp Gly Asp Asn Val Gly Glu Gly Gly Lys GlyAsn Glu 50 55 60 Asp Gly Asn Asp Asp Gln Pro Lys Glu His Ala Ala Gly Asn65 70 75 <210> SEQ ID NO 41 <211> LENGTH: 169 <212> TYPE: PRT <213>ORGANISM: Leishmania major <400> SEQUENCE: 41 Leu Gln Gln Arg Leu AspThr Ala Thr Gln Gln Arg Ala Glu Leu Glu 1 5 10 15 Ala Arg Val Ala ArgLeu Ala Ala Asp Arg Asp Glu Ala Arg Gln Gln 20 25 30 Leu Ala Ala Asn AlaGlu Glu Leu Gln Gln Arg Leu Asp Thr Ala Thr 35 40 45 Gln Gln Arg Ala GluLeu Glu Ala Arg Val Ala Arg Leu Ala Ala Asp 50 55 60 Gly Asp Glu Ala ArgGln Gln Leu Ala Ala Asn Ala Glu Glu Leu Gln 65 70 75 80 Gln Arg Leu AspThr Ala Thr Gln Gln Arg Ala Glu Leu Glu Ala Gln 85 90 95 Val Ala Arg LeuAla Ala Asn Ala Glu Glu Leu Gln Gln Arg Leu Asp 100 105 110 Thr Ala ThrGln Gln Arg Ala Glu Leu Glu Ala Arg Val Ala Arg Leu 115 120 125 Ala AlaAsp Arg Asp Glu Ala Arg Gln Gln Leu Ala Ala Asn Ala Glu 130 135 140 GluLeu Gln Gln Arg Leu Asp Thr Ala Thr Gln Gln Arg Ala Glu Leu 145 150 155160 Glu Ala Gln Val Ala Arg Leu Ala Ala 165 <210> SEQ ID NO 42 <211>LENGTH: 98 <212> TYPE: PRT <213> ORGANISM: Leishmania major <220>FEATURE: <221> NAME/KEY: VARIANT <222> LOCATION: (1)...(98) <223> OTHERINFORMATION: Xaa = Any Amino Acid <400> SEQUENCE: 42 Ala Arg Xaa Ile PheVal Lys Thr Leu Thr Gly Xaa Thr Ile Ala Leu 1 5 10 15 Glu Val Glu ProSer Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln 20 25 30 Asp Lys Glu GlyIle Pro Pro Asp Gln Gln Arg Leu Ile Phe Ala Gly 35 40 45 Lys Gln Leu GluXaa Gly Arg Thr Leu Ser Asp Tyr Asn Ile Gln Lys 50 55 60 Glu Ser Thr LeuHis Leu Val Leu Arg Leu Arg Gly Gly Met Xaa Ile 65 70 75 80 Phe Val LysThr Leu Thr Gly Xaa Thr Ile Ala Leu Glu Val Glu Pro 85 90 95 Asn Asp<210> SEQ ID NO 43 <211> LENGTH: 39 <212> TYPE: PRT <213> ORGANISM:Leishmania major <400> SEQUENCE: 43 Leu Gln Gln Arg Leu Asp Thr Ala ThrGln Gln Arg Ala Glu Leu Glu 1 5 10 15 Ala Arg Val Ala Arg Leu Ala AlaAsp Arg Asp Glu Ala Arg Gln Gln 20 25 30 Leu Ala Ala Asn Ala Glu Glu 35<210> SEQ ID NO 44 <211> LENGTH: 600 <212> TYPE: DNA <213> ORGANISM:Leishmania chagasi <220> FEATURE: <221> NAME/KEY: misc_feature <222>LOCATION: (1)...(600) <223> OTHER INFORMATION: n = A,T,C or G <400>SEQUENCE: 44 cggccgcctc agcgaggagg agatcgagcg catggtgcgc gaggctgccgagttcgagga 60 tgaggaccgc aaggtgcgcg aacgtgtcga agcgaagaac tcgctagagagcatcgcgta 120 ctcgcttcgc aaccagatca acgacaagga caagcttggt gacaagctcgccgcggacga 180 caagaaggcg atcgaggagg ctgtgaagga tgccctcgac tttgtccacgagaaccccaa 240 tgcagaccgt gaggagttcg aggctgctcg cacgaagctg cagagtgtgacgaaccccat 300 cattcaaaag gtgtaccagg gcgccgccgg ctctggtgca gaagaggcggacgcgatgga 360 tgacttgtta gtcggccgcg tgaaaagaaa aacagggaaa gcgggaacatnccacaanaa 420 ccnaagaaga aagggggtng cgacaccgct cgaacaccga cggcncacatncntcatggg 480 catgctcagc tttcctctcc ccaacaaacc agaaggtttt ctccaaacnccgtctcngcn 540 cccaaaatac ggaaangtta ancgaaaaan ccccttccac caattgnngttcttttgttt 600 <210> SEQ ID NO 45 <211> LENGTH: 1748 <212> TYPE: DNA<213> ORGANISM: Leishmania chagasi <400> SEQUENCE: 45 ctagtggatcccccgggctg caggaattca cggaatacgt acctcctccc ccttcttggt 60 agaagaacaacaacaacgtt caagacgacg ccgcgccttc ttgtaccgca tttgcttctg 120 agcacgttcaatccgtgcct tgcaaacatg gaggcgtaca agaagctgga aacgatcttt 180 acgaaggtctaccgcctgga ccacttcctc ggtctgggca actgggacat gaacacaaac 240 atgccccccaagggcgagga atcacgcggt gaggcgatgg cgatgctctc ggagctccgc 300 tttggcttcatcacggcacc ggaggtgaaa agcctgattg agagtgccac caagggcagc 360 gaggagctgaatgcggtgca gcgcgctaac ttgcgggaga tgaggcgtgc gtggaagagc 420 gccaccgccttgccggctga gtttgtgggc cgcaagatgc gcctcacgac acacgcgcac 480 agcgtgtggcgcgacagccg caaagcaaat gacttcgcca agttcctacc ggtgctcagg 540 gacctggtggcgctcgcccg tgaggagggc tcatacctcg ccgccggcac ctccctctcc 600 ccgtatgaggcgctcatgaa cgagtacgag ccaggaatca cgacacaaaa gctggatgag 660 gtgtacgcaaatgtaaagtc gtggctgccg cagctgctaa aggacattgt gcagaagcag 720 tccggcgagtcggtgattgc gttctcgcat aagttcccgc aggacaagca ggaagcactg 780 tgcaaggaattcatgaagat ctggcacttc gacaccgatg ccggtcgcct cgacgtcagc 840 ccccaccctttcacgggaat gacgaaggag gactgccgac tcacaacaaa ctacatcgaa 900 gacacgtttgttcagagctt gtatggcgtc atccacgaga gtgggcatgg caagtacgag 960 cagaactgtggcccacgcga gcacatcacg cagccggtgt gcaacgcccg ctctcttggc 1020 ctgcatgagagccagagcct ctttgcggag tttcagatcg gccacgcgac gcccttcatc 1080 gactacctcacaactcgcct tcctgagttc ttcgaggcgc agccagcgtt ctcgcaggac 1140 aacatgcgcaagtcgctgca gcaggtgaag ccgggctaca ttcgcgtcga tgccgatgag 1200 gtgtgctaccctctgcacgt gatcctgcgc tacgagatcg agcgcgactt gatggagggc 1260 aaaatggaggtggaagacgt gccgcgcgcg tggaacgcaa agatgcagga gtacttgggt 1320 ctctcaacggagggccgtga cgacgttggg tgcctgcagg acgtgcattg gtccatggtg 1380 cgctcggctactctccgacg tactcgctcg gcgccatgta tgcggcgcag atcatggcga 1440 gcatccgaaaggagctggga gacgacaagg tggatgagtg cctgcgcacc ggtgagctcg 1500 gccccctcctggaaaagcag caggagaaga tctgggatca tgggtgcctg tacgagacgg 1560 acgacctcatgacgcgtgcg acgggcgaga cgctgaaccc cgagtacctg cgccgccacc 1620 tggaggcgcgctacataaac gcctgagtcg cgagcggttg acacacgcgc tcgctagcac 1680 atgacgcgtctttattattc tttgttgtgc attcggaatt ccgcggaatt cgatatcaag 1740 cttatcga1748 <210> SEQ ID NO 46 <211> LENGTH: 560 <212> TYPE: DNA <213>ORGANISM: Leishmania chagasi <220> FEATURE: <221> NAME/KEY: misc_feature<222> LOCATION: (1)...(560) <223> OTHER INFORMATION: n = A,T,C or G<400> SEQUENCE: 46 cggaaggagg atggccatac acagaaaaat gacggcgatggccctaagga ggacggccgt 60 acacagaaaa acgacgacgg tggccctaag gaggacggccatacacagaa aaatgacggc 120 gatggcccta aggaggacgg ccgtacacag aaaaataacggcgatggccc tnaggaggac 180 ggccatacac agaaaaatga cggcgatgcc cctnaggaggacggccgtac acanaaaaat 240 gacggcnatg gccctnagga ggacggccgt acacagaaaaatgacngcca tggcccttag 300 gangacgccg tacacagaaa aatgacgcna tggccctnagggaggacggc catacccana 360 aaaattgacg gcnatngccc ttaggangac ggccgtncccanaaanantg acngcggtng 420 cccttaagga agatgaaaat ctgccaccaa aacnattgggaatgcncagg aaaanaacna 480 anatngaccc cacgtggggg atgganctta cngcnattaanattgttacc attatcnacc 540 naaggacnng ttgccgncaa 560 <210> SEQ ID NO 47<211> LENGTH: 600 <212> TYPE: DNA <213> ORGANISM: Leishmania chagasi<220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (1)...(600)<223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 47 cgtccgagaaacccgtacat gtatgctgct ggtagaaggc gcagagctgg tccctctgat 60 gcacaagcatgaggtcgtac attgcctggt tcgtcatttt ccagagcaca acgagcagcg 120 tcatcatacagcatccaata gccgccagag tgaatgcgat gcgcacacca agtcgaaagt 180 ggtcgaccagtaggggaatg tgaccctggc tggcgtgcaa catgatcgcc acgccagcgg 240 tgggccacaccacaacagag gcgacgaaag agaacatgaa cttgctcacg aagctnacaa 300 taagggcgtcgctngtgatg ctaagaacca cgccnaggta gacggcgaag ancaaactaa 360 acacaagcgtgacgatcccg aaaagaagga tctctgcgga attttcgtga gataganaat 420 gcccgtactggaaaaanaag ccggcaggcg cgcgataacg ctgcaacttg ccgctcctcg 480 cgggcgcgttttcgctcctt ctccgacttg atggcgcngt cngncttgac aaaacggtta 540 agctcctcatgccccagccg attcccagct cacggtccac ttccggccat gcccacggac 600 <210> SEQ IDNO 48 <211> LENGTH: 1053 <212> TYPE: DNA <213> ORGANISM: Leishmaniachagasi <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:(1)...(1053) <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 48gggaaaaaag tggagctcca ccgcggtggc ggccgctcta gaactagtgg atcccccggg 60ctgcaggaat tccgcggaat tccgcggaat tccgcggaat tccgtccgac gcggcacccg 120cacaggggtc gacagtgacg caacctcctc caccactgcg gcctacgacg gcgccggctc 180cgcgccagtg atggttgacg ccaatgtgag ccaccctccg tacgcggggc atgaccaagt 240gtacatgcac gtcggcaagc ccatcgtggg caacaccctc gacggataca acgggtgcgt 300gttcgcctac gggcanacgg gcagcggcaa aaccttcacg atgctcggnt acgcgccgag 360cacgancgac atccgcgctc gcaaagggtc cgtcccctgc ggggccagca gcatggagaa 420cagcactcct cttgacagcg ctgtggagcc gtttgagagc gatgacggcg acgacgtggt 480ggacaagacg gggctggatc cgaacgagct gcaaggcatc atcccgcgcg cgtgcacgga 540cctgttcgat ggtctccgtg cgaagcgcgc caaggactcc gacttcacgt accgcgtgga 600ggtgtcttac tacgagatct acaacgagaa ggtgttcgat ctcatccggc cgcagcgcaa 660cacggacctg aggatacgta actcgcccaa ctccggtcca tttatcgaag gcctgacgtg 720gaagatggtg tccaaggagg aagacgtcgc ccgcgtgatt cgcaagggca tgcaggagcg 780ccacacggct gcgaccaagt tcaacgaccg cagcagccgc agccacgcca tcctcacctt 840caacattgtg cagctgtcga tggacgactc cgacaacgcg ttccagatgc gcagcaagct 900gaacctggtg gaccttgctg ggtcggagcg cactggtgcg gccggagccg agggcaatga 960gttccacgac ggtgtgaaga tcaaccactc gctgacggtg ctggggcgcg tgatcgaccg 1020tctggcggac ctctcgcaga acaagggagg ggg 1053 <210> SEQ ID NO 49 <211>LENGTH: 136 <212> TYPE: PRT <213> ORGANISM: Leishmania chagasi <400>SEQUENCE: 49 Gly Arg Leu Ser Glu Glu Glu Ile Glu Arg Met Val Arg Glu AlaAla 1 5 10 15 Glu Phe Glu Asp Glu Asp Arg Lys Val Arg Glu Arg Val GluAla Lys 20 25 30 Asn Ser Leu Glu Ser Ile Ala Tyr Ser Leu Arg Asn Gln IleAsn Asp 35 40 45 Lys Asp Lys Leu Gly Asp Lys Leu Ala Ala Asp Asp Lys LysAla Ile 50 55 60 Glu Glu Ala Val Lys Asp Ala Leu Asp Phe Val His Glu AsnPro Asn 65 70 75 80 Ala Asp Arg Glu Glu Phe Glu Ala Ala Arg Thr Lys LeuGln Ser Val 85 90 95 Thr Asn Pro Ile Ile Gln Lys Val Tyr Gln Gly Ala AlaGly Ser Gly 100 105 110 Ala Glu Glu Ala Asp Ala Met Asp Asp Leu Leu ValGly Arg Val Lys 115 120 125 Arg Lys Thr Gly Lys Ala Gly Thr 130 135<210> SEQ ID NO 50 <211> LENGTH: 510 <212> TYPE: PRT <213> ORGANISM:Leishmania chagasi <400> SEQUENCE: 50 Tyr Leu Leu Pro Leu Leu Gly ArgArg Thr Thr Thr Thr Phe Lys Thr 1 5 10 15 Thr Pro Arg Leu Leu Val ProHis Leu Leu Leu Ser Thr Phe Asn Pro 20 25 30 Cys Leu Ala Asn Met Glu AlaTyr Lys Lys Leu Glu Thr Ile Phe Thr 35 40 45 Lys Val Tyr Arg Leu Asp HisPhe Leu Gly Leu Gly Asn Trp Asp Met 50 55 60 Asn Thr Asn Met Pro Pro LysGly Glu Glu Ser Arg Gly Glu Ala Met 65 70 75 80 Ala Met Leu Ser Glu LeuArg Phe Gly Phe Ile Thr Ala Pro Glu Val 85 90 95 Lys Ser Leu Ile Glu SerAla Thr Lys Gly Ser Glu Glu Leu Asn Ala 100 105 110 Val Gln Arg Ala AsnLeu Arg Glu Met Arg Arg Ala Trp Lys Ser Ala 115 120 125 Thr Ala Leu ProAla Glu Phe Val Gly Arg Lys Met Arg Leu Thr Thr 130 135 140 His Ala HisSer Val Trp Arg Asp Ser Arg Lys Ala Asn Asp Phe Ala 145 150 155 160 LysPhe Leu Pro Val Leu Arg Asp Leu Val Ala Leu Ala Arg Glu Glu 165 170 175Gly Ser Tyr Leu Ala Ala Gly Thr Ser Leu Ser Pro Tyr Glu Ala Leu 180 185190 Met Asn Glu Tyr Glu Pro Gly Ile Thr Thr Gln Lys Leu Asp Glu Val 195200 205 Tyr Ala Asn Val Lys Ser Trp Leu Pro Gln Leu Leu Lys Asp Ile Val210 215 220 Gln Lys Gln Ser Gly Glu Ser Val Ile Ala Phe Ser His Lys PhePro 225 230 235 240 Gln Asp Lys Gln Glu Ala Leu Cys Lys Glu Phe Met LysIle Trp His 245 250 255 Phe Asp Thr Asp Ala Gly Arg Leu Asp Val Ser ProHis Pro Phe Thr 260 265 270 Gly Met Thr Lys Glu Asp Cys Arg Leu Thr ThrAsn Tyr Ile Glu Asp 275 280 285 Thr Phe Val Gln Ser Leu Tyr Gly Val IleHis Glu Ser Gly His Gly 290 295 300 Lys Tyr Glu Gln Asn Cys Gly Pro ArgGlu His Ile Thr Gln Pro Val 305 310 315 320 Cys Asn Ala Arg Ser Leu GlyLeu His Glu Ser Gln Ser Leu Phe Ala 325 330 335 Glu Phe Gln Ile Gly HisAla Thr Pro Phe Ile Asp Tyr Leu Thr Thr 340 345 350 Arg Leu Pro Glu PhePhe Glu Ala Gln Pro Ala Phe Ser Gln Asp Asn 355 360 365 Met Arg Lys SerLeu Gln Gln Val Lys Pro Gly Tyr Ile Arg Val Asp 370 375 380 Ala Asp GluVal Cys Tyr Pro Leu His Val Ile Leu Arg Tyr Glu Ile 385 390 395 400 GluArg Asp Leu Met Glu Gly Lys Met Glu Val Glu Asp Val Pro Arg 405 410 415Ala Trp Asn Ala Lys Met Gln Glu Tyr Leu Gly Leu Ser Thr Glu Gly 420 425430 Arg Asp Asp Val Gly Cys Leu Gln Asp Val His Trp Ser Met Val Arg 435440 445 Ser Ala Thr Leu Arg Arg Thr Arg Ser Ala Pro Cys Met Arg Arg Arg450 455 460 Ser Trp Arg Ala Ser Glu Arg Ser Trp Glu Thr Thr Arg Trp MetSer 465 470 475 480 Ala Cys Ala Pro Val Ser Ser Ala Pro Ser Trp Lys SerSer Arg Arg 485 490 495 Arg Ser Gly Ile Met Gly Ala Cys Thr Arg Arg ThrThr Ser 500 505 510 <210> SEQ ID NO 51 <211> LENGTH: 107 <212> TYPE: PRT<213> ORGANISM: Leishmania chagasi <220> FEATURE: <221> NAME/KEY:VARIANT <222> LOCATION: (1)...(107) <223> OTHER INFORMATION: Xaa = AnyAmino Acid <400> SEQUENCE: 51 Gly Arg Arg Met Ala Ile His Arg Lys MetThr Ala Met Ala Leu Arg 1 5 10 15 Arg Thr Ala Val His Arg Lys Thr ThrThr Val Ala Leu Arg Arg Thr 20 25 30 Ala Ile His Arg Lys Met Thr Ala MetAla Leu Arg Arg Thr Ala Val 35 40 45 His Arg Lys Ile Thr Ala Met Ala LeuArg Arg Thr Ala Ile His Arg 50 55 60 Lys Met Thr Ala Met Pro Leu Arg ArgThr Ala Val His Xaa Lys Met 65 70 75 80 Thr Ala Met Ala Leu Arg Arg ThrAla Val His Arg Lys Met Thr Ala 85 90 95 Met Ala Leu Arg Xaa Thr Pro TyrThr Glu Lys 100 105 <210> SEQ ID NO 52 <211> LENGTH: 63 <212> TYPE: PRT<213> ORGANISM: Leishmania chagasi <400> SEQUENCE: 52 Val Arg Glu ThrArg Thr Cys Met Leu Leu Val Glu Gly Ala Glu Leu 1 5 10 15 Val Pro LeuMet His Lys His Glu Val Val His Cys Leu Val Arg His 20 25 30 Phe Pro GluHis Asn Glu Gln Arg His His Thr Ala Ser Asn Ser Arg 35 40 45 Gln Ser GluCys Asp Ala His Thr Lys Ser Lys Val Val Asp Gln 50 55 60 <210> SEQ ID NO53 <211> LENGTH: 324 <212> TYPE: PRT <213> ORGANISM: Leishmania chagasi<220> FEATURE: <221> NAME/KEY: VARIANT <222> LOCATION: (1)...(324) <223>OTHER INFORMATION: Xaa = Any Amino Acid <400> SEQUENCE: 53 Phe Arg GlyIle Pro Arg Asn Ser Val Arg Arg Gly Thr Arg Thr Gly 1 5 10 15 Val AspSer Asp Ala Thr Ser Ser Thr Thr Ala Ala Tyr Asp Gly Ala 20 25 30 Gly SerAla Pro Val Met Val Asp Ala Asn Val Ser His Pro Pro Tyr 35 40 45 Ala GlyHis Asp Gln Val Tyr Met His Val Gly Lys Pro Ile Val Gly 50 55 60 Asn ThrLeu Asp Gly Tyr Asn Gly Cys Val Phe Ala Tyr Gly Xaa Thr 65 70 75 80 GlySer Gly Lys Thr Phe Thr Met Leu Gly Tyr Ala Pro Ser Thr Xaa 85 90 95 AspIle Arg Ala Arg Lys Gly Ser Val Pro Cys Gly Ala Ser Ser Met 100 105 110Glu Asn Ser Thr Pro Leu Asp Ser Ala Val Glu Pro Phe Glu Ser Asp 115 120125 Asp Gly Asp Asp Val Val Asp Lys Thr Gly Leu Asp Pro Asn Glu Leu 130135 140 Gln Gly Ile Ile Pro Arg Ala Cys Thr Asp Leu Phe Asp Gly Leu Arg145 150 155 160 Ala Lys Arg Ala Lys Asp Ser Asp Phe Thr Tyr Arg Val GluVal Ser 165 170 175 Tyr Tyr Glu Ile Tyr Asn Glu Lys Val Phe Asp Leu IleArg Pro Gln 180 185 190 Arg Asn Thr Asp Leu Arg Ile Arg Asn Ser Pro AsnSer Gly Pro Phe 195 200 205 Ile Glu Gly Leu Thr Trp Lys Met Val Ser LysGlu Glu Asp Val Ala 210 215 220 Arg Val Ile Arg Lys Gly Met Gln Glu ArgHis Thr Ala Ala Thr Lys 225 230 235 240 Phe Asn Asp Arg Ser Ser Arg SerHis Ala Ile Leu Thr Phe Asn Ile 245 250 255 Val Gln Leu Ser Met Asp AspSer Asp Asn Ala Phe Gln Met Arg Ser 260 265 270 Lys Leu Asn Leu Val AspLeu Ala Gly Ser Glu Arg Thr Gly Ala Ala 275 280 285 Gly Ala Glu Gly AsnGlu Phe His Asp Gly Val Lys Ile Asn His Ser 290 295 300 Leu Thr Val LeuGly Arg Val Ile Asp Arg Leu Ala Asp Leu Ser Gln 305 310 315 320 Asn LysGly Gly <210> SEQ ID NO 54 <211> LENGTH: 1585 <212> TYPE: DNA <213>ORGANISM: Leishmania major <220> FEATURE: <221> NAME/KEY: misc_feature<222> LOCATION: (1)...(1585) <223> OTHER INFORMATION: n = A,T,C or G<400> SEQUENCE: 54 aaagctggag ctccaccgcg gtggcggccg ctctagaactagtggatccc ccgggctgca 60 ggaattcggc acgagtgctg cccgacatga catgctcgctgaccggactt cagtgcacag 120 acccgaactg caagacctgc acaacttacg gtcagtgcacagactgcaac gacggctacg 180 gtctcacctc ctccagcgtt tgcgtgcgct gcagtgtagcgggctgcaag agctgccccg 240 tcgacgctaa cgtctgcaaa gtgtgtctcg gcggcagcgagccgatcaac aatatgtgcc 300 cctgcaccga ccccaactgc gccagctgcc ccagcgacgctggcacgtgc actcagtgcg 360 cgaacggcta cggtctcgtg gacggcgcct gtgtgagatgccaggagccc aactgcttca 420 gctgcgacag cgacgcgaat aagtgcacac aatgtgcgccgaactactac ctcaccccgc 480 tcttgacctg ctccccggtg gcctgcaaca tcgagcactgcatgcagtgc gacccacaga 540 cgccgtcgcg ctgccaggag tgcgtgtccc cctacgtggttgacagctac gacggcctct 600 gcaggctctc cgatgcctgc tccgtgccca actgcaagaagtgcgagacc ggtacctcca 660 ggctctgcgc cgagtgcgac accggctaca gtctctccgccgacgcgacg agctgcagca 720 gtccaaccac gcagccgtgc gaggtggagc actgcaacacatgtgtgaac ggcgatagca 780 cccgctgtgc ctactgcaac accggctact acgtctccgatggcaagtgc aaggccatgc 840 agggctgcta cgtgtcgaac tgcgcgcagt gcatgctgcttgacagcacc aagtgctcca 900 cgtgcgtgaa agggtacctg ctcacgtcgt cctacagttgcgtctcgcag aaagtcatca 960 acagtgcggc cgcgccctac tctctgtggg tggccgccgccgtgctcctc acctcttttg 1020 ccatgcacct agcatagtgc gcagcggcat gcgaacaaccccactctcat tctccaacat 1080 gtgcatacac acacacacag acagcggggc agcaccccctccccacacac acacacgcac 1140 ttcccccttg tcttgttctt ctttcctcgn ttcgcatttctttctctcgt gcgctggcgc 1200 cggcctcctg cacgtcgctc ccctccccct aacctctattctctctctct ctctctctcg 1260 ccggcatcat tgcttcttac ccttttctga tccttgctcgcgtgggcgga cactgccaca 1320 gtcccacagc gcagacacac gtgtttaaac ggcgcaggcatccctcccta tcacttcatt 1380 tctcctaaag ccactcacca agtcgcacac cgccctcccccatcggccgc ccttccgggc 1440 gcagctgtgc ggaatgggtg tgtgctcgac ctcgttcctggcagctcact cgcatgtgta 1500 cagccactcc aaccacgaaa gctctcttct gcgcacataaaaaaaaaaaa aaaaaaaaaa 1560 ctcgaggggg ggcccggtac ccaaa 1585 <210> SEQ IDNO 55 <211> LENGTH: 320 <212> TYPE: PRT <213> ORGANISM: Leishmania major<400> SEQUENCE: 55 Val Leu Pro Asp Met Thr Cys Ser Leu Thr Gly Leu GlnCys Thr Asp 1 5 10 15 Pro Asn Cys Lys Thr Cys Thr Thr Tyr Gly Gln CysThr Asp Cys Asn 20 25 30 Asp Gly Tyr Gly Leu Thr Ser Ser Ser Val Cys ValArg Cys Ser Val 35 40 45 Ala Gly Cys Lys Ser Cys Pro Val Asp Ala Asn ValCys Lys Val Cys 50 55 60 Leu Gly Gly Ser Glu Pro Ile Asn Asn Met Cys ProCys Thr Asp Pro 65 70 75 80 Asn Cys Ala Ser Cys Pro Ser Asp Ala Gly ThrCys Thr Gln Cys Ala 85 90 95 Asn Gly Tyr Gly Leu Val Asp Gly Ala Cys ValArg Cys Gln Glu Pro 100 105 110 Asn Cys Phe Ser Cys Asp Ser Asp Ala AsnLys Cys Thr Gln Cys Ala 115 120 125 Pro Asn Tyr Tyr Leu Thr Pro Leu LeuThr Cys Ser Pro Val Ala Cys 130 135 140 Asn Ile Glu His Cys Met Gln CysAsp Pro Gln Thr Pro Ser Arg Cys 145 150 155 160 Gln Glu Cys Val Ser ProTyr Val Val Asp Ser Tyr Asp Gly Leu Cys 165 170 175 Arg Leu Ser Asp AlaCys Ser Val Pro Asn Cys Lys Lys Cys Glu Thr 180 185 190 Gly Thr Ser ArgLeu Cys Ala Glu Cys Asp Thr Gly Tyr Ser Leu Ser 195 200 205 Ala Asp AlaThr Ser Cys Ser Ser Pro Thr Thr Gln Pro Cys Glu Val 210 215 220 Glu HisCys Asn Thr Cys Val Asn Gly Asp Ser Thr Arg Cys Ala Tyr 225 230 235 240Cys Asn Thr Gly Tyr Tyr Val Ser Asp Gly Lys Cys Lys Ala Met Gln 245 250255 Gly Cys Tyr Val Ser Asn Cys Ala Gln Cys Met Leu Leu Asp Ser Thr 260265 270 Lys Cys Ser Thr Cys Val Lys Gly Tyr Leu Leu Thr Ser Ser Tyr Ser275 280 285 Cys Val Ser Gln Lys Val Ile Asn Ser Ala Ala Ala Pro Tyr SerLeu 290 295 300 Trp Val Ala Ala Ala Val Leu Leu Thr Ser Phe Ala Met HisLeu Ala 305 310 315 320 <210> SEQ ID NO 56 <211> LENGTH: 14 <212> TYPE:PRT <213> ORGANISM: Leishmania major <400> SEQUENCE: 56 Pro Lys Glu AspGly His Ala Pro Lys Asn Asp Asp His Ala 1 5 10 <210> SEQ ID NO 57 <211>LENGTH: 7 <212> TYPE: PRT <213> ORGANISM: Leishmania major <400>SEQUENCE: 57 Pro Lys Glu Asp Gly His Ala 1 5 <210> SEQ ID NO 58 <211>LENGTH: 7 <212> TYPE: PRT <213> ORGANISM: Leishmania major <400>SEQUENCE: 58 Pro Lys Asn Asp Asp His Ala 1 5 <210> SEQ ID NO 59 <211>LENGTH: 264 <212> TYPE: DNA <213> ORGANISM: Leishmania chagasi <400>SEQUENCE: 59 atgcaccatc atcaccatca catgggaagc tcctgcacga aggactccgcaaaggagccc 60 cagaagcgtg ctgataacat cgatacgacc actcgaagcg atgagaaggacggcatccat 120 gtccaggaga gcgccggtcc tgtgcaggag aacttcgggg atgcgcaggagaagaacgaa 180 gatggacaca acgtggggga tggagctaac gacaatgagg atggtaacgatgatcagccg 240 aaggagcagg ttgccggcaa ctag 264 <210> SEQ ID NO 60 <211>LENGTH: 744 <212> TYPE: DNA <213> ORGANISM: Leishmania chagasi <400>SEQUENCE: 60 atgggagcct actgcacgaa ggactccgca aaggagcccc agaagcgtgctgataacatc 60 cataaaacca ctgaggccaa tcacagaggc gccgccggtg tgcccccgaagcacgccggc 120 ggtgcgatga acgactctgc cccgaaggag gatggccata cacagaaaaatgacggcgat 180 ggccctaagg aggacggccg tacacagaaa aacgacgacg gtggccctaaggaggacggc 240 catacacaga aaaatgacgg cgatggccct aaggaggacg gccgtacacagaaaaataac 300 ggcgatggcc ctaaggagga cggccataca cagaaaaatg acggcgatgcccctaaggag 360 gacggccgta cacagaaaaa tgacggcgat ggccctaagg aggacggccgtacacagaaa 420 aatgacggcg atggccctaa ggaggacggc cgtacacaga aaaatgacggcgatggccct 480 aaggaggacg gccgtacaca gaaaaatgac ggcgatggcc ctaaggaggacggccataca 540 cagaaaaatg acggcgatgg ccctaaggag gacggccgta cacagaaaaatgacggcggt 600 ggccctaagg aggatgagaa tctgcagcaa aacgatggga atgcgcaggagaagaacgaa 660 gatggacaca acgtggggga tggagctaac ggcaatgagg atggtaacgatgatcagccg 720 aaggagcagg ttgccggcaa ctag 744 <210> SEQ ID NO 61 <211>LENGTH: 80 <212> TYPE: PRT <213> ORGANISM: Leishmania chagasi <400>SEQUENCE: 61 Met Gly Ser Ser Cys Thr Lys Asp Ser Ala Lys Glu Pro Gln LysArg 1 5 10 15 Ala Asp Asn Ile Asp Thr Thr Thr Arg Ser Asp Glu Lys AspGly Ile 20 25 30 His Val Gln Glu Ser Ala Gly Pro Val Gln Glu Asn Phe GlyAsp Ala 35 40 45 Gln Glu Lys Asn Glu Asp Gly His Asn Val Gly Asp Gly AlaAsn Asp 50 55 60 Asn Glu Asp Gly Asn Asp Asp Gln Pro Lys Glu Gln Val AlaGly Asn 65 70 75 80 <210> SEQ ID NO 62 <211> LENGTH: 247 <212> TYPE: PRT<213> ORGANISM: Leishmania chagasi <400> SEQUENCE: 62 Met Gly Ala TyrCys Thr Lys Asp Ser Ala Lys Glu Pro Gln Lys Arg 1 5 10 15 Ala Asp AsnIle His Lys Thr Thr Glu Ala Asn His Arg Gly Ala Ala 20 25 30 Gly Val ProPro Lys His Ala Gly Gly Ala Met Asn Asp Ser Ala Pro 35 40 45 Lys Glu AspGly His Thr Gln Lys Asn Asp Gly Asp Gly Pro Lys Glu 50 55 60 Asp Gly ArgThr Gln Lys Asn Asp Asp Gly Gly Pro Lys Glu Asp Gly 65 70 75 80 His ThrGln Lys Asn Asp Gly Asp Gly Pro Lys Glu Asp Gly Arg Thr 85 90 95 Gln LysAsn Asn Gly Asp Gly Pro Lys Glu Asp Gly His Thr Gln Lys 100 105 110 AsnAsp Gly Asp Ala Pro Lys Glu Asp Gly Arg Thr Gln Lys Asn Asp 115 120 125Gly Asp Gly Pro Lys Glu Asp Gly Arg Thr Gln Lys Asn Asp Gly Asp 130 135140 Gly Pro Lys Glu Asp Gly Arg Thr Gln Lys Asn Asp Gly Asp Gly Pro 145150 155 160 Lys Glu Asp Gly Arg Thr Gln Lys Asn Asp Gly Asp Gly Pro LysGlu 165 170 175 Asp Gly His Thr Gln Lys Asn Asp Gly Asp Gly Pro Lys GluAsp Gly 180 185 190 Arg Thr Gln Lys Asn Asp Gly Gly Gly Pro Lys Glu AspGlu Asn Leu 195 200 205 Gln Gln Asn Asp Gly Asn Ala Gln Glu Lys Asn GluAsp Gly His Asn 210 215 220 Val Gly Asp Gly Ala Asn Gly Asn Glu Asp GlyAsn Asp Asp Gln Pro 225 230 235 240 Lys Glu Gln Val Ala Gly Asn 245<210> SEQ ID NO 63 <211> LENGTH: 14 <212> TYPE: PRT <213> ORGANISM:Leishmania chagasi <220> FEATURE: <221> NAME/KEY: VARIANT <222>LOCATION: (6)...(6) <223> OTHER INFORMATION: Xaa = His or Arg <221>NAME/KEY: VARIANT <222> LOCATION: (12)...(12) <223> OTHER INFORMATION:Xaa = Gly or Asp <221> NAME/KEY: VARIANT <222> LOCATION: (13)...(13)<223> OTHER INFORMATION: Xaa = Asp or Gly <400> SEQUENCE: 63 Pro Lys GluAsp Gly Xaa Thr Gln Lys Asn Asp Xaa Xaa Gly 1 5 10 <210> SEQ ID NO 64<211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM: Leishmania chagasi <220>FEATURE: <221> NAME/KEY: VARIANT <222> LOCATION: (6)...(6) <223> OTHERINFORMATION: Xaa = His or Arg <400> SEQUENCE: 64 Pro Lys Glu Asp Gly XaaThr 1 5 <210> SEQ ID NO 65 <211> LENGTH: 7 <212> TYPE: PRT <213>ORGANISM: Leishmania chagasi <220> FEATURE: <221> NAME/KEY: VARIANT<222> LOCATION: (5)...(5) <223> OTHER INFORMATION: Xaa = Gly or Asp<221> NAME/KEY: VARIANT <222> LOCATION: (6)...(6) <223> OTHERINFORMATION: Xaa = Asp or Gly <400> SEQUENCE: 65 Gln Lys Asn Asp Xaa XaaGly 1 5 <210> SEQ ID NO 66 <211> LENGTH: 17 <212> TYPE: PRT <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Synthetic peptide to asses diagnostic potential of repeat in Lc Gene B<400> SEQUENCE: 66 Gly Cys Gly Pro Lys Glu Asp Gly Arg Thr Gln Lys AsnAsp Gly Asp 1 5 10 15 Gly <210> SEQ ID NO 67 <211> LENGTH: 31 <212>TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Synthetic peptide to asses diagnostic potential of repeatin Lc Gene B <400> SEQUENCE: 67 Gly Cys Gly Pro Lys Glu Asp Gly Arg ThrGln Lys Asn Asp Gly Asp 1 5 10 15 Gly Pro Lys Glu Asp Gly Arg Thr GlnLys Asn Asp Gly Asp Gly 20 25 30 <210> SEQ ID NO 68 <211> LENGTH: 45<212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: Synthetic peptide to asses diagnostic potential ofrepeat in Lc Gene B <400> SEQUENCE: 68 Gly Cys Gly Pro Lys Glu Asp GlyArg Thr Gln Lys Asn Asp Gly Asp 1 5 10 15 Gly Pro Lys Glu Asp Gly ArgThr Gln Lys Asn Asp Gly Asp Gly Pro 20 25 30 Lys Glu Asp Gly Arg Thr GlnLys Asn Asp Gly Asp Gly 35 40 45 <210> SEQ ID NO 69 <211> LENGTH: 17<212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: Synthetic peptide to asses diagnostic potential ofrepeat in Lc Gene B <400> SEQUENCE: 69 Gly Cys Gly Pro Lys Glu Asp GlyHis Thr Gln Lys Asn Asp Gly Asp 1 5 10 15 Gly <210> SEQ ID NO 70 <211>LENGTH: 31 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Synthetic peptide to asses diagnosticpotential of repeat in Lc Gene B <400> SEQUENCE: 70 Gly Cys Gly Pro LysGlu Asp Gly His Thr Gln Lys Asn Asp Gly Asp 1 5 10 15 Gly Pro Lys GluAsp Gly His Thr Gln Lys Asn Asp Gly Asp Gly 20 25 30 <210> SEQ ID NO 71<211> LENGTH: 45 <212> TYPE: PRT <213> ORGANISM: Gly Cys Gly Pro Lys GluAsp Gly His Thr Gln <220> FEATURE: <223> OTHER INFORMATION: Syntheticpeptide to asses diagnostic potential of repeat in Lc Gene B <400>SEQUENCE: 71 Gly Cys Gly Pro Lys Glu Asp Gly His Thr Gln Lys Asn Asp GlyAsp 1 5 10 15 Gly Pro Lys Glu Asp Gly His Thr Gln Lys Asn Asp Gly AspGly Pro 20 25 30 Lys Glu Asp Gly His Thr Gln Lys Asn Asp Gly Asp Gly 3540 45 <210> SEQ ID NO 72 <211> LENGTH: 664 <212> TYPE: DNA <213>ORGANISM: Leishmania major <220> FEATURE: <221> NAME/KEY: misc_feature<222> LOCATION: (1)...(664) <223> OTHER INFORMATION: n = A,T,C or G<400> SEQUENCE: 72 gctgcaggaa ttcggcacga gattgcttcc cagcccaccttcgctatcca gccactctcg 60 ctcttctaca tctcccaccc cctcacaccg ccatggcttcttcccgcaag gcttccaacc 120 cgcacaagtc gcaccgcaag ccgaagcgct cgtggaacgtgtacgtgggc cgctcgctga 180 aggcgatcaa cgcccagatg tcgatgtcgc accgcacgatgaagatcgtg aactcgtacg 240 tgaacgacgt gatggagcgc atctgcactg aggccgcgtcgattgttcgc gcgaacaaga 300 agcgcacgtt gggtgcgcgc gaggtgcaga cggcggtgcgcattgtgctg ccggcggagc 360 tcgcgaagca tgccatggct gagggcacga aggccgtgtcgagcgcgtcc cgctaaagcg 420 gcttgccgga tgccgtgtga gtaggagggt ggcttgccgcaaacgctgac ctcggcgatt 480 gcggcgtggc gctccccttc tcctccttgt ccggcggtgtgtgtcatgca tttgcgtgac 540 tcctccctct tatagatgca agcttttttt ttctcttgacgttttatttt ctcctccccc 600 tcccttaacg tgaagtgtat atganagcgt actggacatgananaaaaaa aaaanaaact 660 cgag 664 <210> SEQ ID NO 73 <211> LENGTH: 1432<212> TYPE: DNA <213> ORGANISM: Leishmania major <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (1)...(1432) <223> OTHERINFORMATION: n = A,T,C or G <400> SEQUENCE: 73 gatgaagaag aggaggacaccaccatcaac aactccgacg tggtggtgcg ctacaagaag 60 gccgcaacgt ggtgcaatgaaacgttgcgc gtgcttatcg atgccacaaa acctggcgcc 120 aaggtgtgcg acctgtgccgcctcggtgat gacaccatca ccgccnaggt caagacaatg 180 ttcaaaggca cggaaaaaggcatcgctttc ccgacctgca tctcggtcaa caactgcgta 240 tgccacaaca gccctggcgtgtcggacgag acgacgcagc aagagatcgc gatgggtgac 300 gtcgtgcact acgacctgggcatccacgtg gacggctact gcgccgtcgt cgcgcacacc 360 attcaggtga cagaggacaatgagcttggc aaggacgaga aggcggcgcg cgtcattaca 420 gcggcgtaca acatcctgaacacggcgctg cgccagatgc gtcccggtac gaccatctac 480 caggtgacag acgtagttgagaaggctgcg gagcactaca aggtgactcc ggtagacggc 540 gtcctctcgc atatgatgaagcgctacatc atagacngat accgctgtat cccgcagcgc 600 agggtcgcgg agcacatggtgcacgactac gatctcgaga aagcgcaggt gtggacgcta 660 gacattgtca tgacctccggcaagggcaag ctgaaggagc gcgatgcgcg gccgtgcgtg 720 ttcaaggtgg ctctggactccaactactct gtgaaaatgg aaagcgcgaa ggaggttcag 780 aaggaaatcg actccnagtatgccaccttc ccctttgcca tccgcaacct ggaggccaag 840 aaggcccgcc tcggtctcaacgagatggcg aagcacggtg ctgtcatccc gtaccctatt 900 ctcttcgaaa aggaaggcgaggtcgtcgcc catttcaaga ttacggtgct catcagcaac 960 aagaagattg agccgattaccggcctgaag ccgcagaagg ccccggcgct cgagccatac 1020 acggacgaga tgctgcttgcgacgaacaag ctcttcgctg tcgctagaga agaaggcggc 1080 gaagtagacg gccgtggcatccgtgacgct gtactgcgag ctttcgtagg cgtacgcctc 1140 ttgtgaggcg tacacgtgtgctgtttgcgg acgaggaggc acccattctg ttccccttct 1200 tcgctaatct tcgcgtttcctctgacgctg gcttctytgc cggagtgtgg tgaggcgcgt 1260 gggggagaaa cggcccactygcatgcctgt gcatacgcga gcacggtagg gagcgcggtg 1320 tgtgtgtgtg tgggggggcgtgttacgagt acaaaagagg ctcgatcttt gcgatctttt 1380 ctttctgtaa acaggaacataagtaaccaa aaaaaaaaaa aaaaaactcg ag 1432 <210> SEQ ID NO 74 <211>LENGTH: 873 <212> TYPE: DNA <213> ORGANISM: Leishmania major <220>FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (1)...(873) <223>OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 74 ctttattgtcatcactgtaa agcactgttt tttctttcac tttttcttga gtgttttctt 60 tattcaccatgagcattat caaggaggac gacgccgtgg gctgctacat gacggtgacc 120 ctcgtggacgacaccaaggt ggagggtacc atcttcacct acaattccaa ggagggcatc 180 atagtactcctgtccctccg cgacgatcag acgaacatga agctaatccg cactccgtac 240 atcaaagacttcagcctttc acacgctgag gagggagcgc acctgccccc ggcactggac 300 tccttcaacgagcttccgtc catgcacgcc ggccgcgaca agtccatctt caagcacgcc 360 agcacgcagctcaagaacgc cgaggcgaac cgcgaaaagc acttcaactc tgtcacgacc 420 gacacaccgattgccacact tgatgcgtac ctcaagctcc tgcggctata ccccttaatt 480 gagtggaacagcgacgaggg tgtcatccag gtctcggaca ccgtcattgt cgtaggagac 540 cccgactggcggacgcccaa ggcaatgctg gtggacggcg cccctgagaa ggacagaccg 600 cttgtagatcgcctgcaggt tgcgctcggm aacggcaaga agtgattcag tgtgtagcgg 660 acagaacatcgtgtgcttgt gtgtctgttt gangtttgtt tgttttctct ttgtggtact 720 gcgtacgacggcgccttctc ccggtggtgg gtgagtccat aagcagttga gttctyggtt 780 gtagnaavgcctyacygccg accatatggg agagggcgaa caaatntttg atagaagttg 840 aaaatcccaaagtyaaaaga aaaaaaaaan aaa 873 <210> SEQ ID NO 75 <211> LENGTH: 1238<212> TYPE: DNA <213> ORGANISM: Leishmania major <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (1)...(1238) <223> OTHERINFORMATION: n = A,T,C or G <400> SEQUENCE: 75 tttctgtact ttattgaacatcagtagaac acgttcttcc cgcaaagatg gccaagaagc 60 acctcaagcg cttgtatgcgcccaaggact ggatgctgag caagctgacc ggcgtgttcg 120 cgccgcgtcc gcgtccgggtccgcacaagc tgcgcgagtg cctgccgctn ctggtgatca 180 tccgcaaccg gctgaagtacgcgctgaacg cgcgcgaggg tgagatgatc ctgcgccagg 240 gtctggtgca cgtggacaaccacccgcgcc gcgacggcaa gtatcccgcc ggtttcatgg 300 acgtggtcga gatcccgaagacgggcgacc gcttccgcct gatgtacgac gtcaagggcc 360 gcttcgcgtt ggtgaacctgtccgaggcgg aggcgcagat caagctgatg aaggttgtga 420 acctgtacac ggccaccggccgcgtgccgg tcgctgtgac gcacgacggc caccgcatcc 480 gctacccgga cccgcacacctccattggtg acaccatcgt gtacaacgtc aaggagaaga 540 agtgcgtgga cctgatcaagaaccgccagg gcaaggccgt gatcgtgacc ggtggcgcca 600 accgcggccg catcggcgagatcgtgaagg tggagtgcca ccccggtgcg ttcaacattg 660 cgcacctgaa ggacgcgtccggcgccgagt tcgccacccg cgccgcgaac atcttcgtga 720 tcggcaagga cctgaacaacctgcaggtaa cggtgccgaa gcagcagggc ctgcgcatga 780 acgtgatcca ggagcgcgaggagcgcctga tcgcggcgga ggcccgcaag aacgcgccgg 840 ctcgtggtgc ccgcagggcccgcaagtgag gaggcgatta cacgcatgcg tgtttgtggc 900 tctgaagcga cttggcgggtcggctgtgag ggtttgagag gaggtgtgtg atgcgtgtga 960 agtccttctc cgttctcagctctctctgtg ctgtagctgt gcctttcccc agatcgcttt 1020 accgcatttg catacatctgtgtagtcgca tgtgcgtgtt tctgtctctc ggtgggtctc 1080 cctctccctc cctttctgcctctctctttg agtgggtgtg catgcgtcgc gcgcgacggg 1140 ctccgcttna gtgattctctcgtgttttan ggctgtttty tttctyagtt nagcgtttty 1200 gttcatgatt tcctcagacccaaaaaaaaa aaaaaaaa 1238 210> SEQ ID NO 76 <211> LENGTH: 712 <212> TYPE:DNA <213> ORGANISM: Leishmania major <220> FEATURE: <221> NAME/KEY:misc_feature <222> LOCATION: (1)...(712) <223> OTHER INFORMATION: n =A,T,C or G <400> SEQUENCE: 76 ctgacggagt tccagacgaa ccttgtgccgtacccgcgca tccacttcgt gctgacaagc 60 tacgctccgg tggtgtctgc cgagaaggcgtaccacgagc agctntccgt cgcggacatc 120 acgaactcgg tntttgagcc tgctggcatgctnacaaagt gcgatcctcg ccacggcaag 180 tacatgtcgt gctgcctcat gtaccgcggtgatgtcgtgc cgaaggatgt caacgccgcg 240 attgcgacga tcaagacgaa gcgcacaattcagttcgtgg actggtgccc gaccggcttc 300 aagtgcggca tcaactacca gccgccgaccgttgtgcccg gcggtgacct cgcgaaggtg 360 cagcgcgccg tgtgcatgat tgccaactcgaccgcgatcg ctgaggtgtt tgcccgcatc 420 gaccacaagt tcgacctgat gtacagcaagcgcgcgtttg tgcactggta cgtgggtgag 480 ggcatggagg agggcgagtt ctccgaggcgcgcgaggatc tcgctgcgct ggagaaggac 540 tacgaggagg ttggcgccga gtccgccgacgacatgggcg aggaggacgt cgaggagtac 600 taaggtagac tcgtgccgcg cgctgatgatgtaggtgcac gcgtgcgtgt gctgcagcgg 660 agccgccgcc accgcgactg tgtgtgtgtgcgcgcgtgac gaccggctcg ag 712 <210> SEQ ID NO 77 <211> LENGTH: 1086 <212>TYPE: DNA <213> ORGANISM: Leishmania major <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (1)...(1086) <223> OTHERINFORMATION: n = A,T,C or G <400> SEQUENCE: 77 caagaagtgg atcaagcaggagacgaacgc cgatggcgag cgcgtgcgcc gcgcgttctg 60 ccagttctgc ctagaccccatctaccagat cttcgacgct gtgatgaacg agaagaagga 120 caaggtggac aagatgctcaagtcgctgca cgtgacgctn acggctgagg agcgcgagca 180 ggtgccgaan aagcttctgaagacggtgat gatgaanttc ctgccggctg ctgagacgct 240 gctacagatg atcgtggcgcacctgccgtc gcccaagaag gcgcaggcgt accgtgcgga 300 gatgctgtac tctggcgaggcgtcgccgga ggacaagtac ttcatgggta tcaagaactg 360 cgaccccgct gcgccgctcatgctgtacat cagcaagatg gtgccgacgg ccgaccgcgg 420 ccgcttcttc gcctttggccgcatcttctc cggtaaggtg cgcagcggcc agaaggtgcg 480 catcatgggt aacaactacgtctacggcaa gaagcaggac ctgtacgagg acaagcctgt 540 gcagcgctcc gtgctgatgatgggccgcta ccaggaggcc gtggaggaca tgccgtgcgg 600 taacgtggtg ggccttgtgggcgtggacaa gtacatcgtg aagtccgcga cgatcacgga 660 cgatggcgag agcccgcacccgctgcgcga catgaagtac tctgtgtcgc ccgtcgtgcg 720 tgtggccgtg gaggcgaagaacccgtccga cctgccgaag cttgtggagg gcctgaagcg 780 ccttgccaag tccgacccgctggtggtgtg cagcattgag gagtctggcg agcacattgt 840 tgccggcgct ggcgagcttcaccttgagat ttgcctgaag gatctccagg aggacttcat 900 gaacggcgcg ccgctnaagatctccgagcc ggtggtgtcg ttccgcgaga cggtgacgga 960 tgtgtcgtcg cagcagtgcctgtcgaagtc tgcgaacaag cacaaccgtc tcttctgccg 1020 cggtgcgccg ctnacagagganctggcgct ggcgatngan gaaggcaccg ctggtcccga 1080 ngcgga 1086 <210> SEQID NO 78 <211> LENGTH: 447 <212> TYPE: DNA <213> ORGANISM: Leishmaniamajor <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:(1)...(447) <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 78cgcatcaacg tctacttcga tnagtcgacg ggaggccgct acgtgccgcg cgccgtgctg 60atggacctcg agcccggcac tatggactcc gttcgcgccg gcccgtacgg ccagctgttc 120cgcccggaca acttcatctt tggtcagtcc ggcgctggca acaactgggc caagggccac 180tacactgagg gcgcggagct gatcgactcc gtgcttgatg tgtgccgcaa ggaggcggag 240agctgcgact gcctgcaggg cttccagctg tctcactccc tcggcggcgg cacgggctcc 300ggcatgggca cgctgctcat ttccaanctg cgcgangagt acccggaccg gatcatgatg 360accttctccg tcatcccgtc cccccgcgtg tcggataccg ttgtggancc gtacaacacg 420accctctctg tgcaccagct cgtggaa 447 <210> SEQ ID NO 79 <211> LENGTH: 375<212> TYPE: DNA <213> ORGANISM: Leishmania major <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (1)...(375) <223> OTHERINFORMATION: n = A,T,C or G <400> SEQUENCE: 79 gtaacccgct ggtgtacgcatatgtagaca cagacgggca gcacgagacg acgttcctcg 60 cgatccctgt ggtgcttggcatgaatggaa tcgagaagcg cctgccgatt ggtccgctgc 120 actcgacgga ggaaacgctgctgaaggcgg cactgccggt gatcaagaag aatatcgtga 180 agggcagcga gttcgcgcgctcacacctgt agcacctcag cttttttttt ttgcgttaaa 240 cgggcgtggg aagcacctcgatacttcgct tcgcgctgac ggacccgcac gacatcgttc 300 gtcatccccc tccccctcttcggccctata cgcatgaagg agtggaatta tgcaacagca 360 tgttnatatc aagtg 375<210> SEQ ID NO 80 <211> LENGTH: 107 <212> TYPE: PRT <213> ORGANISM:Leishmania major <400> SEQUENCE: 80 Met Ala Ser Ser Arg Lys Ala Ser AsnPro His Lys Ser His Arg Lys 1 5 10 15 Pro Lys Arg Ser Trp Asn Val TyrVal Gly Arg Ser Leu Lys Ala Ile 20 25 30 Asn Ala Gln Met Ser Met Ser HisArg Thr Met Lys Ile Val Asn Ser 35 40 45 Tyr Val Asn Asp Val Met Glu ArgIle Cys Thr Glu Ala Ala Ser Ile 50 55 60 Val Arg Ala Asn Lys Lys Arg ThrLeu Gly Ala Arg Glu Val Gln Thr 65 70 75 80 Ala Val Arg Ile Val Leu ProAla Glu Leu Ala Lys His Ala Met Ala 85 90 95 Glu Gly Thr Lys Ala Val SerSer Ala Ser Arg 100 105 <210> SEQ ID NO 81 <211> LENGTH: 381 <212> TYPE:PRT <213> ORGANISM: Leishmania major <220> FEATURE: <221> NAME/KEY:VARIANT <222> LOCATION: (1)...(381) <223> OTHER INFORMATION: Xaa = AnyAmino Acid <400> SEQUENCE: 81 Asp Glu Glu Glu Glu Asp Thr Thr Ile AsnAsn Ser Asp Val Val Val 1 5 10 15 Arg Tyr Lys Lys Ala Ala Thr Trp CysAsn Glu Thr Leu Arg Val Leu 20 25 30 Ile Asp Ala Thr Lys Pro Gly Ala LysVal Cys Asp Leu Cys Arg Leu 35 40 45 Gly Asp Asp Thr Ile Thr Ala Xaa ValLys Thr Met Phe Lys Gly Thr 50 55 60 Glu Lys Gly Ile Ala Phe Pro Thr CysIle Ser Val Asn Asn Cys Val 65 70 75 80 Cys His Asn Ser Pro Gly Val SerAsp Glu Thr Thr Gln Gln Glu Ile 85 90 95 Ala Met Gly Asp Val Val His TyrAsp Leu Gly Ile His Val Asp Gly 100 105 110 Tyr Cys Ala Val Val Ala HisThr Ile Gln Val Thr Glu Asp Asn Glu 115 120 125 Leu Gly Lys Asp Glu LysAla Ala Arg Val Ile Thr Ala Ala Tyr Asn 130 135 140 Ile Leu Asn Thr AlaLeu Arg Gln Met Arg Pro Gly Thr Thr Ile Tyr 145 150 155 160 Gln Val ThrAsp Val Val Glu Lys Ala Ala Glu His Tyr Lys Val Thr 165 170 175 Pro ValAsp Gly Val Leu Ser His Met Met Lys Arg Tyr Ile Ile Asp 180 185 190 XaaTyr Arg Cys Ile Pro Gln Arg Arg Val Ala Glu His Met Val His 195 200 205Asp Tyr Asp Leu Glu Lys Ala Gln Val Trp Thr Leu Asp Ile Val Met 210 215220 Thr Ser Gly Lys Gly Lys Leu Lys Glu Arg Asp Ala Arg Pro Cys Val 225230 235 240 Phe Lys Val Ala Leu Asp Ser Asn Tyr Ser Val Lys Met Glu SerAla 245 250 255 Lys Glu Val Gln Lys Glu Ile Asp Ser Xaa Tyr Ala Thr PhePro Phe 260 265 270 Ala Ile Arg Asn Leu Glu Ala Lys Lys Ala Arg Leu GlyLeu Asn Glu 275 280 285 Met Ala Lys His Gly Ala Val Ile Pro Tyr Pro IleLeu Phe Glu Lys 290 295 300 Glu Gly Glu Val Val Ala His Phe Lys Ile ThrVal Leu Ile Ser Asn 305 310 315 320 Lys Lys Ile Glu Pro Ile Thr Gly LeuLys Pro Gln Lys Ala Pro Ala 325 330 335 Leu Glu Pro Tyr Thr Asp Glu MetLeu Leu Ala Thr Asn Lys Leu Phe 340 345 350 Ala Val Ala Arg Glu Glu GlyGly Glu Val Asp Gly Arg Gly Ile Arg 355 360 365 Asp Ala Val Leu Arg AlaPhe Val Gly Val Arg Leu Leu 370 375 380 <210> SEQ ID NO 82 <211> LENGTH:191 <212> TYPE: PRT <213> ORGANISM: Leishmania major <400> SEQUENCE: 82Met Ser Ile Ile Lys Glu Asp Asp Ala Val Gly Cys Tyr Met Thr Val 1 5 1015 Thr Leu Val Asp Asp Thr Lys Val Glu Gly Thr Ile Phe Thr Tyr Asn 20 2530 Ser Lys Glu Gly Ile Ile Val Leu Leu Ser Leu Arg Asp Asp Gln Thr 35 4045 Asn Met Lys Leu Ile Arg Thr Pro Tyr Ile Lys Asp Phe Ser Leu Ser 50 5560 His Ala Glu Glu Gly Ala His Leu Pro Pro Ala Leu Asp Ser Phe Asn 65 7075 80 Glu Leu Pro Ser Met His Ala Gly Arg Asp Lys Ser Ile Phe Lys His 8590 95 Ala Ser Thr Gln Leu Lys Asn Ala Glu Ala Asn Arg Glu Lys His Phe100 105 110 Asn Ser Val Thr Thr Asp Thr Pro Ile Ala Thr Leu Asp Ala TyrLeu 115 120 125 Lys Leu Leu Arg Leu Tyr Pro Leu Ile Glu Trp Asn Ser AspGlu Gly 130 135 140 Val Ile Gln Val Ser Asp Thr Val Ile Val Val Gly AspPro Asp Trp 145 150 155 160 Arg Thr Pro Lys Ala Met Leu Val Asp Gly AlaPro Glu Lys Asp Arg 165 170 175 Pro Leu Val Asp Arg Leu Gln Val Ala LeuGly Asn Gly Lys Lys 180 185 190 <210> SEQ ID NO 83 <211> LENGTH: 273<212> TYPE: PRT <213> ORGANISM: Leishmania major <400> SEQUENCE: 83 MetAla Lys Lys His Leu Lys Arg Leu Tyr Ala Pro Lys Asp Trp Met 1 5 10 15Leu Ser Lys Leu Thr Gly Val Phe Ala Pro Arg Pro Arg Pro Gly Pro 20 25 30His Lys Leu Arg Glu Cys Leu Pro Leu Leu Val Ile Ile Arg Asn Arg 35 40 45Leu Lys Tyr Ala Leu Asn Ala Arg Glu Gly Glu Met Ile Leu Arg Gln 50 55 60Gly Leu Val His Val Asp Asn His Pro Arg Arg Asp Gly Lys Tyr Pro 65 70 7580 Ala Gly Phe Met Asp Val Val Glu Ile Pro Lys Thr Gly Asp Arg Phe 85 9095 Arg Leu Met Tyr Asp Val Lys Gly Arg Phe Ala Leu Val Asn Leu Ser 100105 110 Glu Ala Glu Ala Gln Ile Lys Leu Met Lys Val Val Asn Leu Tyr Thr115 120 125 Ala Thr Gly Arg Val Pro Val Ala Val Thr His Asp Gly His ArgIle 130 135 140 Arg Tyr Pro Asp Pro His Thr Ser Ile Gly Asp Thr Ile ValTyr Asn 145 150 155 160 Val Lys Glu Lys Lys Cys Val Asp Leu Ile Lys AsnArg Gln Gly Lys 165 170 175 Ala Val Ile Val Thr Gly Gly Ala Asn Arg GlyArg Ile Gly Glu Ile 180 185 190 Val Lys Val Glu Cys His Pro Gly Ala PheAsn Ile Ala His Leu Lys 195 200 205 Asp Ala Ser Gly Ala Glu Phe Ala ThrArg Ala Ala Asn Ile Phe Val 210 215 220 Ile Gly Lys Asp Leu Asn Asn LeuGln Val Thr Val Pro Lys Gln Gln 225 230 235 240 Gly Leu Arg Met Asn ValIle Gln Glu Arg Glu Glu Arg Leu Ile Ala 245 250 255 Ala Glu Ala Arg LysAsn Ala Pro Ala Arg Gly Ala Arg Arg Ala Arg 260 265 270 Lys <210> SEQ IDNO 84 <211> LENGTH: 200 <212> TYPE: PRT <213> ORGANISM: Leishmania major<400> SEQUENCE: 84 Leu Thr Glu Phe Gln Thr Asn Leu Val Pro Tyr Pro ArgIle His Phe 1 5 10 15 Val Leu Thr Ser Tyr Ala Pro Val Val Ser Ala GluLys Ala Tyr His 20 25 30 Glu Gln Leu Ser Val Ala Asp Ile Thr Asn Ser ValPhe Glu Pro Ala 35 40 45 Gly Met Leu Thr Lys Cys Asp Pro Arg His Gly LysTyr Met Ser Cys 50 55 60 Cys Leu Met Tyr Arg Gly Asp Val Val Pro Lys AspVal Asn Ala Ala 65 70 75 80 Ile Ala Thr Ile Lys Thr Lys Arg Thr Ile GlnPhe Val Asp Trp Cys 85 90 95 Pro Thr Gly Phe Lys Cys Gly Ile Asn Tyr GlnPro Pro Thr Val Val 100 105 110 Pro Gly Gly Asp Leu Ala Lys Val Gln ArgAla Val Cys Met Ile Ala 115 120 125 Asn Ser Thr Ala Ile Ala Glu Val PheAla Arg Ile Asp His Lys Phe 130 135 140 Asp Leu Met Tyr Ser Lys Arg AlaPhe Val His Trp Tyr Val Gly Glu 145 150 155 160 Gly Met Glu Glu Gly GluPhe Ser Glu Ala Arg Glu Asp Leu Ala Ala 165 170 175 Leu Glu Lys Asp TyrGlu Glu Val Gly Ala Glu Ser Ala Asp Asp Met 180 185 190 Gly Glu Glu AspVal Glu Glu Tyr 195 200 <210> SEQ ID NO 85 <211> LENGTH: 361 <212> TYPE:PRT <213> ORGANISM: Leishmania major <220> FEATURE: <221> NAME/KEY:VARIANT <222> LOCATION: (1)...(361) <223> OTHER INFORMATION: Xaa = AnyAmino Acid <400> SEQUENCE: 85 Lys Lys Trp Ile Lys Gln Glu Thr Asn AlaAsp Gly Glu Arg Val Arg 1 5 10 15 Arg Ala Phe Cys Gln Phe Cys Leu AspPro Ile Tyr Gln Ile Phe Asp 20 25 30 Ala Val Met Asn Glu Lys Lys Asp LysVal Asp Lys Met Leu Lys Ser 35 40 45 Leu His Val Thr Leu Thr Ala Glu GluArg Glu Gln Val Pro Xaa Lys 50 55 60 Leu Leu Lys Thr Val Met Met Xaa PheLeu Pro Ala Ala Glu Thr Leu 65 70 75 80 Leu Gln Met Ile Val Ala His LeuPro Ser Pro Lys Lys Ala Gln Ala 85 90 95 Tyr Arg Ala Glu Met Leu Tyr SerGly Glu Ala Ser Pro Glu Asp Lys 100 105 110 Tyr Phe Met Gly Ile Lys AsnCys Asp Pro Ala Ala Pro Leu Met Leu 115 120 125 Tyr Ile Ser Lys Met ValPro Thr Ala Asp Arg Gly Arg Phe Phe Ala 130 135 140 Phe Gly Arg Ile PheSer Gly Lys Val Arg Ser Gly Gln Lys Val Arg 145 150 155 160 Ile Met GlyAsn Asn Tyr Val Tyr Gly Lys Lys Gln Asp Leu Tyr Glu 165 170 175 Asp LysPro Val Gln Arg Ser Val Leu Met Met Gly Arg Tyr Gln Glu 180 185 190 AlaVal Glu Asp Met Pro Cys Gly Asn Val Val Gly Leu Val Gly Val 195 200 205Asp Lys Tyr Ile Val Lys Ser Ala Thr Ile Thr Asp Asp Gly Glu Ser 210 215220 Pro His Pro Leu Arg Asp Met Lys Tyr Ser Val Ser Pro Val Val Arg 225230 235 240 Val Ala Val Glu Ala Lys Asn Pro Ser Asp Leu Pro Lys Leu ValGlu 245 250 255 Gly Leu Lys Arg Leu Ala Lys Ser Asp Pro Leu Val Val CysSer Ile 260 265 270 Glu Glu Ser Gly Glu His Ile Val Ala Gly Ala Gly GluLeu His Leu 275 280 285 Glu Ile Cys Leu Lys Asp Leu Gln Glu Asp Phe MetAsn Gly Ala Pro 290 295 300 Leu Lys Ile Ser Glu Pro Val Val Ser Phe ArgGlu Thr Val Thr Asp 305 310 315 320 Val Ser Ser Gln Gln Cys Leu Ser LysSer Ala Asn Lys His Asn Arg 325 330 335 Leu Phe Cys Arg Gly Ala Pro LeuThr Glu Xaa Leu Ala Leu Ala Xaa 340 345 350 Xaa Glu Gly Thr Ala Gly ProXaa Ala 355 360 <210> SEQ ID NO 86 <211> LENGTH: 149 <212> TYPE: PRT<213> ORGANISM: Leishmania major <220> FEATURE: <221> NAME/KEY: VARIANT<222> LOCATION: (1)...(149) <223> OTHER INFORMATION: Xaa = Any AminoAcid <400> SEQUENCE: 86 Arg Ile Asn Val Tyr Phe Asp Xaa Ser Thr Gly GlyArg Tyr Val Pro 1 5 10 15 Arg Ala Val Leu Met Asp Leu Glu Pro Gly ThrMet Asp Ser Val Arg 20 25 30 Ala Gly Pro Tyr Gly Gln Leu Phe Arg Pro AspAsn Phe Ile Phe Gly 35 40 45 Gln Ser Gly Ala Gly Asn Asn Trp Ala Lys GlyHis Tyr Thr Glu Gly 50 55 60 Ala Glu Leu Ile Asp Ser Val Leu Asp Val CysArg Lys Glu Ala Glu 65 70 75 80 Ser Cys Asp Cys Leu Gln Gly Phe Gln LeuSer His Ser Leu Gly Gly 85 90 95 Gly Thr Gly Ser Gly Met Gly Thr Leu LeuIle Ser Xaa Leu Arg Xaa 100 105 110 Glu Tyr Pro Asp Arg Ile Met Met ThrPhe Ser Val Ile Pro Ser Pro 115 120 125 Arg Val Ser Asp Thr Val Val XaaPro Tyr Asn Thr Thr Leu Ser Val 130 135 140 His Gln Leu Val Glu 145<210> SEQ ID NO 87 <211> LENGTH: 69 <212> TYPE: PRT <213> ORGANISM:Leishmania major <400> SEQUENCE: 87 Asn Pro Leu Val Tyr Ala Tyr Val AspThr Asp Gly Gln His Glu Thr 1 5 10 15 Thr Phe Leu Ala Ile Pro Val ValLeu Gly Met Asn Gly Ile Glu Lys 20 25 30 Arg Leu Pro Ile Gly Pro Leu HisSer Thr Glu Glu Thr Leu Leu Lys 35 40 45 Ala Ala Leu Pro Val Ile Lys LysAsn Ile Val Lys Gly Ser Glu Phe 50 55 60 Ala Arg Ser His Leu 65 <210>SEQ ID NO 88 <211> LENGTH: 54 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: PCR primer <400>SEQUENCE: 88 agtattcata tgcaccacca ccaccaccac atgtcctgcg gtaacgccaa gatc54 <210> SEQ ID NO 89 <211> LENGTH: 33 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR primer<400> SEQUENCE: 89 ctcacaggat ccctgcttgc tgaagtatcc ttc 33 <210> SEQ IDNO 90 <211> LENGTH: 36 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: PCR primer <400>SEQUENCE: 90 catttcggat ccatggacgc aactgagctg aagaac 36 <210> SEQ ID NO91 <211> LENGTH: 33 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: PCR primer <400> SEQUENCE: 91cgtagagaat tcctgaccaa aacgaatgat gcc 33 <210> SEQ ID NO 92 <211> LENGTH:33 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: PCR primer <400> SEQUENCE: 92 caccacgaattcatggcgca gaatgataag atc 33 <210> SEQ ID NO 93 <211> LENGTH: 34 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: PCR primer <400> SEQUENCE: 93 actgacctcg aggaattcttagtcgcgcat gaac 34 <210> SEQ ID NO 94 <211> LENGTH: 3012 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: DNA sequence encoding fusion (poly-protein) constructscomprising multiple Leishmania antigens <400> SEQUENCE: 94 catatgcaccaccaccacca ccacatgtcc tgcggtaacg ccaagatcaa ctctcccgcg 60 ccgtccttcgaggaggtggc gctcatgccc aacggcagct tcaagaagat cagcctctcc 120 tcctacaagggcaagtgggt cgtgctcttc ttctacccgc tcgacttcac cttcgtgtgc 180 ccgacagaggtcatcgcgtt ctccgacagc gtgagtcgct tcaacgagct caactgcgag 240 gtcctcgcgtgctcgataga cagcgagtac gcgcacctgc agtggacgct gcaggaccgc 300 aagaagggcggcctcgggac catggcgatc ccaatgctag ccgacaagac caagagcatc 360 gctcgttcctacggcgtgct ggaggagagc cagggcgtgg cctaccgcgg tctcttcatc 420 atcgacccccatggcatgct gcgtcagatc accgtcaatg acatgccggt gggccgcagc 480 gtggaggaggttctacgcct gctggaggct tttcagttcg tggagaagca cggcgaggtg 540 tgccccgcgaactggaagaa gggcgccccc acgatgaagc cggaaccgaa tgcgtctgtc 600 gagggatacttcagcaagca gggatccatg gacgcaactg agctgaagaa caaggggaac 660 gaagagttctccgccggccg ctatgtggag gcggtgaact acttctcaaa ggcgatccag 720 ttggatgagcagaacagtgt cctctacagc aaccgctccg cctgttttgc agccatgcag 780 aaatacaaggacgcgctgga cgacgccgac aagtgcatct cgatcaagcc gaattgggcc 840 aagggctacgtgcgccgagg agcagctctc catggcatgc gccgctacga cgatgccatt 900 gccgcgtatgaaaaggggct caaggtggac ccttccaaca gcggctgcgc gcagggcgtg 960 aaggacgtgcaggtagccaa ggcccgcgaa gcacgtgacc ccatcgctcg cgtcttcacc 1020 ccggaggcgttccgcaagat ccaagagaat cccaagctgt ctctacttat gctgcagccg 1080 gactacgtgaagatggtaga caccgtcatc cgcgaccctt cgcagggccg gctgtacatg 1140 gaagaccagcgctttgccct gacgctcatg tacctgagcg gaatgaagat tcccaacgat 1200 ggtgatggcgaggaggagga acgtccgtct gcgaaggcgg cagagacagc gaagccaaaa 1260 gaggagaagcctctcaccga caacgagaag gaggccctgg cgctcaagga ggagggcaac 1320 aagctgtacctctcgaagaa gtttgaggag gcgctgacca agtaccaaga ggcgcaggtg 1380 aaagaccccaacaacacttt atacattctg aacgtgtcgg ccgtgtactt cgagcagggt 1440 gactacgacaagtgcatcgc cgagtgcgag cacggtatcg agcacggtcg cgagaaccac 1500 tgcgactacacaatcattgc gaagctcatg acccggaacg ccttgtgcct ccagaggcag 1560 aggaagtacgaggctgctat cgacctttac aagcgcgccc ttgtcgagtg gcgtaaccct 1620 gacaccctcaagaagctgac ggagtgcgag aaggagcacc aaaaggcggt ggaggaagcc 1680 tacatcgatcctgagatcgc gaagcagaag aaagacgaag gtaaccagta cttcaaggag 1740 gataagttccccgaggccgt ggcagcgtac acggaggcca tcaagcgcaa ccctgccgag 1800 cacacctcctacagcaatcg cgcggccgcg tacatcaagc ttggagcctt caacgacgcc 1860 ctcaaggacgcggagaagtg cattgagctg aagcccgact ttgttaaggg ctacgcgcgc 1920 aagggtcatgcttacttttg gaccaagcag tacaaccgcg cgctgcaggc gtacaatgag 1980 ggcctcaaggtggacccgag caatgcggac tgcaaggatg ggcggtatcg cacaatcatg 2040 aagattcaggagatggcatc tggccaatcc gcggatggcg acgaggcggc gcgccgggcc 2100 atggacgatcctgaaatcgc ggcaatcatg caagatagct acatgcaact agtgttgaag 2160 gagatgcagaacgatcccac gcgcattcag gagtacatga aggactccgg gatctcatcg 2220 aagatcaacaagctgatttc agctggcatc attcgttttg gtcaggaatt catggcgcag 2280 aatgataagatcgcccccca ggaccaggac tccttcctcg atgaccagcc cggcgttcgc 2340 ccgatcccgtccttcgacga catgccgctg caccagaacc tgctgcgtgg catctactcg 2400 tacgggttcgagaagccgtc cagcatccag cagcgcgcga tagccccctt cacgcgcggc 2460 ggcgacatcatcgcgcaggc ccagtccggt accggcaaga cgggtgcctt ctccatcggt 2520 ctgctgcagcgcctggactt ccgccacaac ctgatccagg gcctcgtgct ctcccccact 2580 cgcgagctggccctgcagac ggcggaggtg atcagccgca tcggtgagtt cctgtcgaac 2640 agctccaagttctgcgagac ctttgtcggc ggcacgcgcg tgcaggatga cctgcgcaag 2700 ctgcaggccggcgtcatcgt tgccgtgggc acgccgggcc gcgtgtccga cgtgatcaag 2760 cgtggcgcgctgcgcacaga gtcgctgcgc gtgctggtgc tcgacgaggc tgatgagatg 2820 ctgtctcagggcttcgcgga ccagatttac gagatcttcc gcttcctgcc gaaggacatc 2880 caggtcgcgctcttctccgc cacgatgccg gaggaggtac tggagctgac gaagaagttc 2940 atgcgcgactaagaattcct cgagcagatc cggctgctaa caaagcccga aaggaagctg 3000 aatggctgctgc 3012 <210> SEQ ID NO 95 <211> LENGTH: 982 <212> TYPE: PRT <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Fusion (poly-protein) constructs comprising multiple Leishmania antigens<400> SEQUENCE: 95 Met His His His His His His Met Ser Cys Gly Asn AlaLys Ile Asn 5 10 15 Ser Pro Ala Pro Ser Phe Glu Glu Val Ala Leu Met ProAsn Gly Ser 20 25 30 Phe Lys Lys Ile Ser Leu Ser Ser Tyr Lys Gly Lys TrpVal Val Leu 35 40 45 Phe Phe Tyr Pro Leu Asp Phe Thr Phe Val Cys Pro ThrGlu Val Ile 50 55 60 Ala Phe Ser Asp Ser Val Ser Arg Phe Asn Glu Leu AsnCys Glu Val 65 70 75 80 Leu Ala Cys Ser Ile Asp Ser Glu Tyr Ala His LeuGln Trp Thr Leu 85 90 95 Gln Asp Arg Lys Lys Gly Gly Leu Gly Thr Met AlaIle Pro Met Leu 100 105 110 Ala Asp Lys Thr Lys Ser Ile Ala Arg Ser TyrGly Val Leu Glu Glu 115 120 125 Ser Gln Gly Val Ala Tyr Arg Gly Leu PheIle Ile Asp Pro His Gly 130 135 140 Met Leu Arg Gln Ile Thr Val Asn AspMet Pro Val Gly Arg Ser Val 145 150 155 160 Glu Glu Val Leu Arg Leu LeuGlu Ala Phe Gln Phe Val Glu Lys His 165 170 175 Gly Glu Val Cys Pro AlaAsn Trp Lys Lys Gly Ala Pro Thr Met Lys 180 185 190 Pro Glu Pro Asn AlaSer Val Glu Gly Tyr Phe Ser Lys Gln Gly Ser 195 200 205 Met Asp Ala ThrGlu Leu Lys Asn Lys Gly Asn Glu Glu Phe Ser Ala 210 215 220 Gly Arg TyrVal Glu Ala Val Asn Tyr Phe Ser Lys Ala Ile Gln Leu 225 230 235 240 AspGlu Gln Asn Ser Val Leu Tyr Ser Asn Arg Ser Ala Cys Phe Ala 245 250 255Ala Met Gln Lys Tyr Lys Asp Ala Leu Asp Asp Ala Asp Lys Cys Ile 260 265270 Ser Ile Lys Pro Asn Trp Ala Lys Gly Tyr Val Arg Arg Gly Ala Ala 275280 285 Leu His Gly Met Arg Arg Tyr Asp Asp Ala Ile Ala Ala Tyr Glu Lys290 295 300 Gly Leu Lys Val Asp Pro Ser Asn Ser Gly Cys Ala Gln Gly ValLys 305 310 315 320 Asp Val Gln Val Ala Lys Ala Arg Glu Ala Arg Asp ProIle Ala Arg 325 330 335 Val Phe Thr Pro Glu Ala Phe Arg Lys Ile Gln GluAsn Pro Lys Leu 340 345 350 Ser Leu Leu Met Leu Gln Pro Asp Tyr Val LysMet Val Asp Thr Val 355 360 365 Ile Arg Asp Pro Ser Gln Gly Arg Leu TyrMet Glu Asp Gln Arg Phe 370 375 380 Ala Leu Thr Leu Met Tyr Leu Ser GlyMet Lys Ile Pro Asn Asp Gly 385 390 395 400 Asp Gly Glu Glu Glu Glu ArgPro Ser Ala Lys Ala Ala Glu Thr Ala 405 410 415 Lys Pro Lys Glu Glu LysPro Leu Thr Asp Asn Glu Lys Glu Ala Leu 420 425 430 Ala Leu Lys Glu GluGly Asn Lys Leu Tyr Leu Ser Lys Lys Phe Glu 435 440 445 Glu Ala Leu ThrLys Tyr Gln Glu Ala Gln Val Lys Asp Pro Asn Asn 450 455 460 Thr Leu TyrIle Leu Asn Val Ser Ala Val Tyr Phe Glu Gln Gly Asp 465 470 475 480 TyrAsp Lys Cys Ile Ala Glu Cys Glu His Gly Ile Glu His Gly Arg 485 490 495Glu Asn His Cys Asp Tyr Thr Ile Ile Ala Lys Leu Met Thr Arg Asn 500 505510 Ala Leu Cys Leu Gln Arg Gln Arg Lys Tyr Glu Ala Ala Ile Asp Leu 515520 525 Tyr Lys Arg Ala Leu Val Glu Trp Arg Asn Pro Asp Thr Leu Lys Lys530 535 540 Leu Thr Glu Cys Glu Lys Glu His Gln Lys Ala Val Glu Glu AlaTyr 545 550 555 560 Ile Asp Pro Glu Ile Ala Lys Gln Lys Lys Asp Glu GlyAsn Gln Tyr 565 570 575 Phe Lys Glu Asp Lys Phe Pro Glu Ala Val Ala AlaTyr Thr Glu Ala 580 585 590 Ile Lys Arg Asn Pro Ala Glu His Thr Ser TyrSer Asn Arg Ala Ala 595 600 605 Ala Tyr Ile Lys Leu Gly Ala Phe Asn AspAla Leu Lys Asp Ala Glu 610 615 620 Lys Cys Ile Glu Leu Lys Pro Asp PheVal Lys Gly Tyr Ala Arg Lys 625 630 635 640 Gly His Ala Tyr Phe Trp ThrLys Gln Tyr Asn Arg Ala Leu Gln Ala 645 650 655 Tyr Asn Glu Gly Leu LysVal Asp Pro Ser Asn Ala Asp Cys Lys Asp 660 665 670 Gly Arg Tyr Arg ThrIle Met Lys Ile Gln Glu Met Ala Ser Gly Gln 675 680 685 Ser Ala Asp GlyAsp Glu Ala Ala Arg Arg Ala Met Asp Asp Pro Glu 690 695 700 Ile Ala AlaIle Met Gln Asp Ser Tyr Met Gln Leu Val Leu Lys Glu 705 710 715 720 MetGln Asn Asp Pro Thr Arg Ile Gln Glu Tyr Met Lys Asp Ser Gly 725 730 735Ile Ser Ser Lys Ile Asn Lys Leu Ile Ser Ala Gly Ile Ile Arg Phe 740 745750 Gly Gln Glu Phe Met Ala Gln Asn Asp Lys Ile Ala Pro Gln Asp Gln 755760 765 Asp Ser Phe Leu Asp Asp Gln Pro Gly Val Arg Pro Ile Pro Ser Phe770 775 780 Asp Asp Met Pro Leu His Gln Asn Leu Leu Arg Gly Ile Tyr SerTyr 785 790 795 800 Gly Phe Glu Lys Pro Ser Ser Ile Gln Gln Arg Ala IleAla Pro Phe 805 810 815 Thr Arg Gly Gly Asp Ile Ile Ala Gln Ala Gln SerGly Thr Gly Lys 820 825 830 Thr Gly Ala Phe Ser Ile Gly Leu Leu Gln ArgLeu Asp Phe Arg His 835 840 845 Asn Leu Ile Gln Gly Leu Val Leu Ser ProThr Arg Glu Leu Ala Leu 850 855 860 Gln Thr Ala Glu Val Ile Ser Arg IleGly Glu Phe Leu Ser Asn Ser 865 870 875 880 Ser Lys Phe Cys Glu Thr PheVal Gly Gly Thr Arg Val Gln Asp Asp 885 890 895 Leu Arg Lys Leu Gln AlaGly Val Ile Val Ala Val Gly Thr Pro Gly 900 905 910 Arg Val Ser Asp ValIle Lys Arg Gly Ala Leu Arg Thr Glu Ser Leu 915 920 925 Arg Val Leu ValLeu Asp Glu Ala Asp Glu Met Leu Ser Gln Gly Phe 930 935 940 Ala Asp GlnIle Tyr Glu Ile Phe Arg Phe Leu Pro Lys Asp Ile Gln 945 950 955 960 ValAla Leu Phe Ser Ala Thr Met Pro Glu Glu Val Leu Glu Leu Thr 965 970 975Lys Lys Phe Met Arg Asp 980 <210> SEQ ID NO 96 <211> LENGTH: 1641 <212>TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Fusion (poly-protein) constructs comprising multipleLeishmania antigens <400> SEQUENCE: 96 Met His His His His His His MetSer Cys Gly Asn Ala Lys Ile Asn 5 10 15 Ser Pro Ala Pro Ser Phe Glu GluVal Ala Leu Met Pro Asn Gly Ser 20 25 30 Phe Lys Lys Ile Ser Leu Ser SerTyr Lys Gly Lys Trp Val Val Leu 35 40 45 Phe Phe Tyr Pro Leu Asp Phe ThrPhe Val Cys Pro Thr Glu Val Ile 50 55 60 Ala Phe Ser Asp Ser Val Ser ArgPhe Asn Glu Leu Asn Cys Glu Val 65 70 75 80 Leu Ala Cys Ser Ile Asp SerGlu Tyr Ala His Leu Gln Trp Thr Leu 85 90 95 Gln Asp Arg Lys Lys Gly GlyLeu Gly Thr Met Ala Ile Pro Met Leu 100 105 110 Ala Asp Lys Thr Lys SerIle Ala Arg Ser Tyr Gly Val Leu Glu Glu 115 120 125 Ser Gln Gly Val AlaTyr Arg Gly Leu Phe Ile Ile Asp Pro His Gly 130 135 140 Met Leu Arg GlnIle Thr Val Asn Asp Met Pro Val Gly Arg Ser Val 145 150 155 160 Glu GluVal Leu Arg Leu Leu Glu Ala Phe Gln Phe Val Glu Lys His 165 170 175 GlyGlu Val Cys Pro Ala Asn Trp Lys Lys Gly Ala Pro Thr Met Lys 180 185 190Pro Glu Pro Asn Ala Ser Val Glu Gly Tyr Phe Ser Lys Gln Gly Ser 195 200205 Met Asp Ala Thr Glu Leu Lys Asn Lys Gly Asn Glu Glu Phe Ser Ala 210215 220 Gly Arg Tyr Val Glu Ala Val Asn Tyr Phe Ser Lys Ala Ile Gln Leu225 230 235 240 Asp Glu Gln Asn Ser Val Leu Tyr Ser Asn Arg Ser Ala CysPhe Ala 245 250 255 Ala Met Gln Lys Tyr Lys Asp Ala Leu Asp Asp Ala AspLys Cys Ile 260 265 270 Ser Ile Lys Pro Asn Trp Ala Lys Gly Tyr Val ArgArg Gly Ala Ala 275 280 285 Leu His Gly Met Arg Arg Tyr Asp Asp Ala IleAla Ala Tyr Glu Lys 290 295 300 Gly Leu Lys Val Asp Pro Ser Asn Ser GlyCys Ala Gln Gly Val Lys 305 310 315 320 Asp Val Gln Val Ala Lys Ala ArgGlu Ala Arg Asp Pro Ile Ala Arg 325 330 335 Val Phe Thr Pro Glu Ala PheArg Lys Ile Gln Glu Asn Pro Lys Leu 340 345 350 Ser Leu Leu Met Leu GlnPro Asp Tyr Val Lys Met Val Asp Thr Val 355 360 365 Ile Arg Asp Pro SerGln Gly Arg Leu Tyr Met Glu Asp Gln Arg Phe 370 375 380 Ala Leu Thr LeuMet Tyr Leu Ser Gly Met Lys Ile Pro Asn Asp Gly 385 390 395 400 Asp GlyGlu Glu Glu Glu Arg Pro Ser Ala Lys Ala Ala Glu Thr Ala 405 410 415 LysPro Lys Glu Glu Lys Pro Leu Thr Asp Asn Glu Lys Glu Ala Leu 420 425 430Ala Leu Lys Glu Glu Gly Asn Lys Leu Tyr Leu Ser Lys Lys Phe Glu 435 440445 Glu Ala Leu Thr Lys Tyr Gln Glu Ala Gln Val Lys Asp Pro Asn Asn 450455 460 Thr Leu Tyr Ile Leu Asn Val Ser Ala Val Tyr Phe Glu Gln Gly Asp465 470 475 480 Tyr Asp Lys Cys Ile Ala Glu Cys Glu His Gly Ile Glu HisGly Arg 485 490 495 Glu Asn His Cys Asp Tyr Thr Ile Ile Ala Lys Leu MetThr Arg Asn 500 505 510 Ala Leu Cys Leu Gln Arg Gln Arg Lys Tyr Glu AlaAla Ile Asp Leu 515 520 525 Tyr Lys Arg Ala Leu Val Glu Trp Arg Asn ProAsp Thr Leu Lys Lys 530 535 540 Leu Thr Glu Cys Glu Lys Glu His Gln LysAla Val Glu Glu Ala Tyr 545 550 555 560 Ile Asp Pro Glu Ile Ala Lys GlnLys Lys Asp Glu Gly Asn Gln Tyr 565 570 575 Phe Lys Glu Asp Lys Phe ProGlu Ala Val Ala Ala Tyr Thr Glu Ala 580 585 590 Ile Lys Arg Asn Pro AlaGlu His Thr Ser Tyr Ser Asn Arg Ala Ala 595 600 605 Ala Tyr Ile Lys LeuGly Ala Phe Asn Asp Ala Leu Lys Asp Ala Glu 610 615 620 Lys Cys Ile GluLeu Lys Pro Asp Phe Val Lys Gly Tyr Ala Arg Lys 625 630 635 640 Gly HisAla Tyr Phe Trp Thr Lys Gln Tyr Asn Arg Ala Leu Gln Ala 645 650 655 TyrAsn Glu Gly Leu Lys Val Asp Pro Ser Asn Ala Asp Cys Lys Asp 660 665 670Gly Arg Tyr Arg Thr Ile Met Lys Ile Gln Glu Met Ala Ser Gly Gln 675 680685 Ser Ala Asp Gly Asp Glu Ala Ala Arg Arg Ala Met Asp Asp Pro Glu 690695 700 Ile Ala Ala Ile Met Gln Asp Ser Tyr Met Gln Leu Val Leu Lys Glu705 710 715 720 Met Gln Asn Asp Pro Thr Arg Ile Gln Glu Tyr Met Lys AspSer Gly 725 730 735 Ile Ser Ser Lys Ile Asn Lys Leu Ile Ser Ala Gly IleIle Arg Phe 740 745 750 Gly Gln Glu Phe Ser Leu Thr Asp Pro Ala Val LeuGly Glu Glu Thr 755 760 765 His Leu Arg Val Arg Val Val Pro Asp Lys AlaAsn Lys Thr Leu Thr 770 775 780 Val Glu Asp Asn Gly Ile Gly Met Thr LysAla Asp Leu Val Asn Asn 785 790 795 800 Leu Gly Thr Ile Ala Arg Ser GlyThr Lys Ala Phe Met Glu Ala Leu 805 810 815 Glu Ala Gly Gly Asp Met SerMet Ile Gly Gln Phe Gly Val Gly Phe 820 825 830 Tyr Ser Ala Tyr Leu ValAla Asp Arg Val Thr Val Val Ser Lys Asn 835 840 845 Asn Ser Asp Glu AlaTyr Val Trp Glu Ser Ser Ala Gly Gly Thr Phe 850 855 860 Thr Ile Thr SerVal Pro Glu Ser Asp Met Lys Arg Gly Thr Arg Ile 865 870 875 880 Thr LeuHis Leu Lys Glu Asp Gln Gln Glu Tyr Leu Glu Glu Arg Arg 885 890 895 ValLys Glu Leu Ile Lys Lys His Ser Glu Phe Ile Gly Tyr Asp Ile 900 905 910Glu Leu Met Val Glu Lys Thr Ala Glu Lys Glu Val Thr Asp Glu Asp 915 920925 Glu Glu Glu Asp Glu Ser Lys Lys Lys Ser Cys Gly Asp Glu Gly Glu 930935 940 Pro Lys Val Glu Glu Val Thr Glu Gly Gly Glu Asp Lys Lys Lys Lys945 950 955 960 Thr Lys Lys Val Lys Glu Val Thr Lys Thr Tyr Glu Val GlnAsn Lys 965 970 975 His Lys Pro Leu Trp Thr Arg Asp Pro Lys Asp Val ThrLys Glu Glu 980 985 990 Tyr Ala Ala Phe Tyr Lys Ala Ile Ser Asn Asp TrpGlu Asp Pro Ala 995 1000 1005 Ala Thr Lys His Phe Ser Val Glu Gly GlnLeu Glu Phe Arg Ala Ile 1010 1015 1020 Ala Phe Val Pro Lys Arg Ala ProPhe Asp Met Phe Glu Pro Asn Lys 1025 1030 1035 1040 Lys Arg Asn Asn IleLys Leu Tyr Val Arg Arg Val Phe Ile Met Asp 1045 1050 1055 Asn Cys GluAsp Leu Cys Pro Asp Trp Leu Gly Phe Val Lys Gly Val 1060 1065 1070 ValAsp Ser Glu Asp Leu Pro Leu Asn Ile Ser Arg Glu Asn Leu Gln 1075 10801085 Gln Asn Lys Ile Leu Lys Val Ile Arg Lys Asn Ile Val Lys Lys Cys1090 1095 1100 Leu Glu Leu Phe Glu Glu Ile Ala Glu Asn Lys Glu Asp TyrLys Gln 1105 1110 1115 1120 Phe Tyr Glu Gln Phe Gly Lys Asn Ile Lys LeuGly Ile His Glu Asp 1125 1130 1135 Thr Ala Asn Arg Lys Lys Leu Met GluLeu Leu Arg Phe Tyr Ser Thr 1140 1145 1150 Glu Ser Gly Glu Glu Met ThrThr Leu Lys Asp Tyr Val Thr Arg Met 1155 1160 1165 Lys Pro Glu Gln LysSer Ile Tyr Tyr Ile Thr Gly Asp Ser Lys Lys 1170 1175 1180 Lys Leu GluSer Ser Pro Phe Ile Glu Lys Ala Arg Arg Cys Gly Leu 1185 1190 1195 1200Glu Val Leu Phe Met Thr Glu Pro Ile Asp Glu Tyr Val Met Gln Gln 12051210 1215 Val Lys Asp Phe Glu Asp Lys Lys Phe Ala Cys Leu Thr Lys GluGly 1220 1225 1230 Val His Phe Glu Glu Ser Glu Glu Glu Lys Lys Gln ArgGlu Glu Lys 1235 1240 1245 Lys Ala Ala Cys Glu Lys Leu Cys Lys Thr MetLys Glu Val Leu Gly 1250 1255 1260 Asp Lys Val Glu Lys Val Thr Val SerGlu Arg Leu Ser Thr Ser Pro 1265 1270 1275 1280 Cys Ile Leu Val Thr SerGlu Phe Gly Trp Ser Ala His Met Glu Gln 1285 1290 1295 Ile Met Arg AsnGln Ala Leu Arg Asp Ser Ser Met Ala Gln Tyr Met 1300 1305 1310 Val SerLys Lys Thr Met Glu Val Asn Pro Asp His Pro Ile Ile Lys 1315 1320 1325Glu Leu Arg Arg Arg Val Glu Ala Asp Glu Asn Asp Lys Ala Val Lys 13301335 1340 Asp Leu Val Phe Leu Leu Phe Asp Thr Ser Leu Leu Thr Ser GlyPhe 1345 1350 1355 1360 Gln Leu Asp Asp Pro Thr Gly Tyr Ala Glu Arg IleAsn Arg Met Ile 1365 1370 1375 Lys Leu Gly Leu Ser Leu Asp Glu Glu GluGlu Glu Val Ala Glu Ala 1380 1385 1390 Pro Pro Ala Glu Ala Ala Pro AlaGlu Val Thr Ala Gly Thr Ser Ser 1395 1400 1405 Met Glu Gln Val Asp AspIle Met Ala Gln Asn Asp Lys Ile Ala Pro 1410 1415 1420 Gln Asp Gln AspSer Phe Leu Asp Asp Gln Pro Gly Val Arg Pro Ile 1425 1430 1435 1440 ProSer Phe Asp Asp Met Pro Leu His Gln Asn Leu Leu Arg Gly Ile 1445 14501455 Tyr Ser Tyr Gly Phe Glu Lys Pro Ser Ser Ile Gln Gln Arg Ala Ile1460 1465 1470 Ala Pro Phe Thr Arg Gly Gly Asp Ile Ile Ala Gln Ala GlnSer Gly 1475 1480 1485 Thr Gly Lys Thr Gly Ala Phe Ser Ile Gly Leu LeuGln Arg Leu Asp 1490 1495 1500 Phe Arg His Asn Leu Ile Gln Gly Leu ValLeu Ser Pro Thr Arg Glu 1505 1510 1515 1520 Leu Ala Leu Gln Thr Ala GluVal Ile Ser Arg Ile Gly Glu Phe Leu 1525 1530 1535 Ser Asn Ser Ser LysPhe Cys Glu Thr Phe Val Gly Gly Thr Arg Val 1540 1545 1550 Gln Asp AspLeu Arg Lys Leu Gln Ala Gly Val Ile Val Ala Val Gly 1555 1560 1565 ThrPro Gly Arg Val Ser Asp Val Ile Lys Arg Gly Ala Leu Arg Thr 1570 15751580 Glu Ser Leu Arg Val Leu Val Leu Asp Glu Ala Asp Glu Met Leu Ser1585 1590 1595 1600 Gln Gly Phe Ala Asp Gln Ile Tyr Glu Ile Phe Arg PheLeu Pro Lys 1605 1610 1615 Asp Ile Gln Val Ala Leu Phe Ser Ala Thr MetPro Glu Glu Val Leu 1620 1625 1630 Glu Leu Thr Lys Lys Phe Met Arg Asp1635 1640 <210> SEQ ID NO 97 <211> LENGTH: 1427 <212> TYPE: PRT <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Fusion (poly-protein) constructs comprising multiple Leishmania antigens<400> SEQUENCE: 97 Met His His His His His His Met Ser Cys Gly Asn AlaLys Ile Asn 5 10 15 Ser Pro Ala Pro Ser Phe Glu Glu Val Ala Leu Met ProAsn Gly Ser 20 25 30 Phe Lys Lys Ile Ser Leu Ser Ser Tyr Lys Gly Lys TrpVal Val Leu 35 40 45 Phe Phe Tyr Pro Leu Asp Phe Thr Phe Val Cys Pro ThrGlu Val Ile 50 55 60 Ala Phe Ser Asp Ser Val Ser Arg Phe Asn Glu Leu AsnCys Glu Val 65 70 75 80 Leu Ala Cys Ser Ile Asp Ser Glu Tyr Ala His LeuGln Trp Thr Leu 85 90 95 Gln Asp Arg Lys Lys Gly Gly Leu Gly Thr Met AlaIle Pro Met Leu 100 105 110 Ala Asp Lys Thr Lys Ser Ile Ala Arg Ser TyrGly Val Leu Glu Glu 115 120 125 Ser Gln Gly Val Ala Tyr Arg Gly Leu PheIle Ile Asp Pro His Gly 130 135 140 Met Leu Arg Gln Ile Thr Val Asn AspMet Pro Val Gly Arg Ser Val 145 150 155 160 Glu Glu Val Leu Arg Leu LeuGlu Ala Phe Gln Phe Val Glu Lys His 165 170 175 Gly Glu Val Cys Pro AlaAsn Trp Lys Lys Gly Ala Pro Thr Met Lys 180 185 190 Pro Glu Pro Asn AlaSer Val Glu Gly Tyr Phe Ser Lys Gln Gly Ser 195 200 205 Met Asp Ala ThrGlu Leu Lys Asn Lys Gly Asn Glu Glu Phe Ser Ala 210 215 220 Gly Arg TyrVal Glu Ala Val Asn Tyr Phe Ser Lys Ala Ile Gln Leu 225 230 235 240 AspGlu Gln Asn Ser Val Leu Tyr Ser Asn Arg Ser Ala Cys Phe Ala 245 250 255Ala Met Gln Lys Tyr Lys Asp Ala Leu Asp Asp Ala Asp Lys Cys Ile 260 265270 Ser Ile Lys Pro Asn Trp Ala Lys Gly Tyr Val Arg Arg Gly Ala Ala 275280 285 Leu His Gly Met Arg Arg Tyr Asp Asp Ala Ile Ala Ala Tyr Glu Lys290 295 300 Gly Leu Lys Val Asp Pro Ser Asn Ser Gly Cys Ala Gln Gly ValLys 305 310 315 320 Asp Val Gln Val Ala Lys Ala Arg Glu Ala Arg Asp ProIle Ala Arg 325 330 335 Val Phe Thr Pro Glu Ala Phe Arg Lys Ile Gln GluAsn Pro Lys Leu 340 345 350 Ser Leu Leu Met Leu Gln Pro Asp Tyr Val LysMet Val Asp Thr Val 355 360 365 Ile Arg Asp Pro Ser Gln Gly Arg Leu TyrMet Glu Asp Gln Arg Phe 370 375 380 Ala Leu Thr Leu Met Tyr Leu Ser GlyMet Lys Ile Pro Asn Asp Gly 385 390 395 400 Asp Gly Glu Glu Glu Glu ArgPro Ser Ala Lys Ala Ala Glu Thr Ala 405 410 415 Lys Pro Lys Glu Glu LysPro Leu Thr Asp Asn Glu Lys Glu Ala Leu 420 425 430 Ala Leu Lys Glu GluGly Asn Lys Leu Tyr Leu Ser Lys Lys Phe Glu 435 440 445 Glu Ala Leu ThrLys Tyr Gln Glu Ala Gln Val Lys Asp Pro Asn Asn 450 455 460 Thr Leu TyrIle Leu Asn Val Ser Ala Val Tyr Phe Glu Gln Gly Asp 465 470 475 480 TyrAsp Lys Cys Ile Ala Glu Cys Glu His Gly Ile Glu His Gly Arg 485 490 495Glu Asn His Cys Asp Tyr Thr Ile Ile Ala Lys Leu Met Thr Arg Asn 500 505510 Ala Leu Cys Leu Gln Arg Gln Arg Lys Tyr Glu Ala Ala Ile Asp Leu 515520 525 Tyr Lys Arg Ala Leu Val Glu Trp Arg Asn Pro Asp Thr Leu Lys Lys530 535 540 Leu Thr Glu Cys Glu Lys Glu His Gln Lys Ala Val Glu Glu AlaTyr 545 550 555 560 Ile Asp Pro Glu Ile Ala Lys Gln Lys Lys Asp Glu GlyAsn Gln Tyr 565 570 575 Phe Lys Glu Asp Lys Phe Pro Glu Ala Val Ala AlaTyr Thr Glu Ala 580 585 590 Ile Lys Arg Asn Pro Ala Glu His Thr Ser TyrSer Asn Arg Ala Ala 595 600 605 Ala Tyr Ile Lys Leu Gly Ala Phe Asn AspAla Leu Lys Asp Ala Glu 610 615 620 Lys Cys Ile Glu Leu Lys Pro Asp PheVal Lys Gly Tyr Ala Arg Lys 625 630 635 640 Gly His Ala Tyr Phe Trp ThrLys Gln Tyr Asn Arg Ala Leu Gln Ala 645 650 655 Tyr Asn Glu Gly Leu LysVal Asp Pro Ser Asn Ala Asp Cys Lys Asp 660 665 670 Gly Arg Tyr Arg ThrIle Met Lys Ile Gln Glu Met Ala Ser Gly Gln 675 680 685 Ser Ala Asp GlyAsp Glu Ala Ala Arg Arg Ala Met Asp Asp Pro Glu 690 695 700 Ile Ala AlaIle Met Gln Asp Ser Tyr Met Gln Leu Val Leu Lys Glu 705 710 715 720 MetGln Asn Asp Pro Thr Arg Ile Gln Glu Tyr Met Lys Asp Ser Gly 725 730 735Ile Ser Ser Lys Ile Asn Lys Leu Ile Ser Ala Gly Ile Ile Arg Phe 740 745750 Gly Gln Glu Phe Ser Leu Thr Asp Pro Ala Val Leu Gly Glu Glu Thr 755760 765 His Leu Arg Val Arg Val Val Pro Asp Lys Ala Asn Lys Thr Leu Thr770 775 780 Val Glu Asp Asn Gly Ile Gly Met Thr Lys Ala Asp Leu Val AsnAsn 785 790 795 800 Leu Gly Thr Ile Ala Arg Ser Gly Thr Lys Ala Phe MetGlu Ala Leu 805 810 815 Glu Ala Gly Gly Asp Met Ser Met Ile Gly Gln PheGly Val Gly Phe 820 825 830 Tyr Ser Ala Tyr Leu Val Ala Asp Arg Val ThrVal Val Ser Lys Asn 835 840 845 Asn Ser Asp Glu Ala Tyr Val Trp Glu SerSer Ala Gly Gly Thr Phe 850 855 860 Thr Ile Thr Ser Val Pro Glu Ser AspMet Lys Arg Gly Thr Arg Ile 865 870 875 880 Thr Leu His Leu Lys Glu AspGln Gln Glu Tyr Leu Glu Glu Arg Arg 885 890 895 Val Lys Glu Leu Ile LysLys His Ser Glu Phe Ile Gly Tyr Asp Ile 900 905 910 Glu Leu Met Val GluLys Thr Ala Glu Lys Glu Val Thr Asp Glu Asp 915 920 925 Glu Glu Glu AspGlu Ser Lys Lys Lys Ser Cys Gly Asp Glu Gly Glu 930 935 940 Pro Lys ValGlu Glu Val Thr Glu Gly Gly Glu Asp Lys Lys Lys Lys 945 950 955 960 ThrLys Lys Val Lys Glu Val Thr Lys Thr Tyr Glu Val Gln Asn Lys 965 970 975His Lys Pro Leu Trp Thr Arg Asp Pro Lys Asp Val Thr Lys Glu Glu 980 985990 Tyr Ala Ala Phe Tyr Lys Ala Ile Ser Asn Asp Trp Glu Asp Pro Ala 9951000 1005 Ala Thr Lys His Phe Ser Val Glu Gly Gln Leu Glu Phe Arg AlaIle 1010 1015 1020 Ala Phe Val Pro Lys Arg Ala Pro Phe Asp Met Phe GluPro Asn Lys 1025 1030 1035 1040 Lys Arg Asn Asn Ile Lys Leu Tyr Val ArgArg Val Phe Ile Met Asp 1045 1050 1055 Asn Cys Glu Asp Leu Cys Pro AspTrp Leu Gly Phe Val Lys Gly Val 1060 1065 1070 Val Asp Ser Glu Asp LeuPro Leu Asn Ile Ser Arg Glu Asn Leu Gln 1075 1080 1085 Gln Asn Lys IleLeu Lys Val Ile Arg Lys Asn Ile Val Lys Lys Cys 1090 1095 1100 Leu GluLeu Phe Glu Glu Ile Ala Glu Asn Lys Glu Asp Tyr Lys Gln 1105 1110 11151120 Phe Tyr Glu Gln Phe Gly Lys Asn Ile Lys Leu Gly Ile His Glu Asp1125 1130 1135 Thr Ala Asn Arg Lys Lys Leu Met Glu Leu Leu Arg Phe TyrSer Thr 1140 1145 1150 Glu Ser Gly Glu Glu Met Thr Thr Leu Lys Asp TyrVal Thr Arg Met 1155 1160 1165 Lys Pro Glu Gln Lys Ser Ile Tyr Tyr IleThr Gly Asp Ser Lys Lys 1170 1175 1180 Lys Leu Glu Ser Ser Pro Phe IleGlu Lys Ala Arg Arg Cys Gly Leu 1185 1190 1195 1200 Glu Val Leu Phe MetThr Glu Pro Ile Asp Glu Tyr Val Met Gln Gln 1205 1210 1215 Val Lys AspPhe Glu Asp Lys Lys Phe Ala Cys Leu Thr Lys Glu Gly 1220 1225 1230 ValHis Phe Glu Glu Ser Glu Glu Glu Lys Lys Gln Arg Glu Glu Lys 1235 12401245 Lys Ala Ala Cys Glu Lys Leu Cys Lys Thr Met Lys Glu Val Leu Gly1250 1255 1260 Asp Lys Val Glu Lys Val Thr Val Ser Glu Arg Leu Ser ThrSer Pro 1265 1270 1275 1280 Cys Ile Leu Val Thr Ser Glu Phe Gly Trp SerAla His Met Glu Gln 1285 1290 1295 Ile Met Arg Asn Gln Ala Leu Arg AspSer Ser Met Ala Gln Tyr Met 1300 1305 1310 Val Ser Lys Lys Thr Met GluVal Asn Pro Asp His Pro Ile Ile Lys 1315 1320 1325 Glu Leu Arg Arg ArgVal Glu Ala Asp Glu Asn Asp Lys Ala Val Lys 1330 1335 1340 Asp Leu ValPhe Leu Leu Phe Asp Thr Ser Leu Leu Thr Ser Gly Phe 1345 1350 1355 1360Gln Leu Asp Asp Pro Thr Gly Tyr Ala Glu Arg Ile Asn Arg Met Ile 13651370 1375 Lys Leu Gly Leu Ser Leu Asp Glu Glu Glu Glu Glu Val Ala GluAla 1380 1385 1390 Pro Pro Ala Glu Ala Ala Pro Ala Glu Val Thr Ala GlyThr Ser Ser 1395 1400 1405 Met Glu Gln Val Asp Asp Ile His His Thr GlyGly Arg Ser Ser Arg 1410 1415 1420 Ser Gly Cys 1425 <210> SEQ ID NO 98<211> LENGTH: 4929 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: DNA sequence encoding fusion(poly-protein) constructs comprising multiple Leishmania antigens <400>SEQUENCE: 98 catatgcacc accaccacca ccacatgtcc tgcggtaacg ccaagatcaactctcccgcg 60 ccgtccttcg aggaggtggc gctcatgccc aacggcagct tcaagaagatcagcctctcc 120 tcctacaagg gcaagtgggt cgtgctcttc ttctacccgc tcgacttcaccttcgtgtgc 180 ccgacagagg tcatcgcgtt ctccgacagc gtgagtcgct tcaacgagctcaactgcgag 240 gtcctcgcgt gctcgataga cagcgagtac gcgcacctgc agtggacgctgcaggaccgc 300 aagaagggcg gcctcgggac catggcgatc ccaatgctag ccgacaagaccaagagcatc 360 gctcgttcct acggcgtgct ggaggagagc cagggcgtgg cctaccgcggtctcttcatc 420 atcgaccccc atggcatgct gcgtcagatc accgtcaatg acatgccggtgggccgcagc 480 gtggaggagg ttctacgcct gctggaggct tttcagttcg tggagaagcacggcgaggtg 540 tgccccgcga actggaagaa gggcgccccc acgatgaagc cggaaccgaatgcgtctgtc 600 gagggatact tcagcaagca gggatccatg gacgcaactg agctgaagaacaaggggaac 660 gaagagttct ccgccggccg ctatgtggag gcggtgaact acttctcaaaggcgatccag 720 ttggatgagc agaacagtgt cctctacagc aaccgctccg cctgttttgcagccatgcag 780 aaatacaagg acgcgctgga cgacgccgac aagtgcatct cgatcaagccgaattgggcc 840 aagggctacg tgcgccgagg agcagctctc catggcatgc gccgctacgacgatgccatt 900 gccgcgtatg aaaaggggct caaggtggac ccttccaaca gcggctgcgcgcagggcgtg 960 aaggacgtgc aggtagccaa ggcccgcgaa gcacgtgacc ccatcgctcgcgtcttcacc 1020 ccggaggcgt tccgcaagat ccaagagaat cccaagctgt ctctacttatgctgcagccg 1080 gactacgtga agatggtaga caccgtcatc cgcgaccctt cgcagggccggctgtacatg 1140 gaagaccagc gctttgccct gacgctcatg tacctgagcg gaatgaagattcccaacgat 1200 ggtgatggcg aggaggagga acgtccgtct gcgaaggcgg cagagacagcgaagccaaaa 1260 gaggagaagc ctctcaccga caacgagaag gaggccctgg cgctcaaggaggagggcaac 1320 aagctgtacc tctcgaagaa gtttgaggag gcgctgacca agtaccaagaggcgcaggtg 1380 aaagacccca acaacacttt atacattctg aacgtgtcgg ccgtgtacttcgagcagggt 1440 gactacgaca agtgcatcgc cgagtgcgag cacggtatcg agcacggtcgcgagaaccac 1500 tgcgactaca caatcattgc gaagctcatg acccggaacg ccttgtgcctccagaggcag 1560 aggaagtacg aggctgctat cgacctttac aagcgcgccc ttgtcgagtggcgtaaccct 1620 gacaccctca agaagctgac ggagtgcgag aaggagcacc aaaaggcggtggaggaagcc 1680 tacatcgatc ctgagatcgc gaagcagaag aaagacgaag gtaaccagtacttcaaggag 1740 gataagttcc ccgaggccgt ggcagcgtac acggaggcca tcaagcgcaaccctgccgag 1800 cacacctcct acagcaatcg cgcggccgcg tacatcaagc ttggagccttcaacgacgcc 1860 ctcaaggacg cggagaagtg cattgagctg aagcccgact ttgttaagggctacgcgcgc 1920 aagggtcatg cttacttttg gaccaagcag tacaaccgcg cgctgcaggcgtacaatgag 1980 ggcctcaagg tggacccgag caatgcggac tgcaaggatg ggcggtatcgcacaatcatg 2040 aagattcagg agatggcatc tggccaatcc gcggatggcg acgaggcggcgcgccgggcc 2100 atggacgatc ctgaaatcgc ggcaatcatg caagatagct acatgcaactagtgttgaag 2160 gagatgcaga acgatcccac gcgcattcag gagtacatga aggactccgggatctcatcg 2220 aagatcaaca agctgatttc agctggcatc attcgttttg gtcaggaattcagcctgacg 2280 gacccggcgg tgctgggcga ggagactcac ctgcgcgtcc gcgtggtgccggacaaggcg 2340 aacaagacgc tgacggtgga ggataacggc atcggcatga ccaaggcggacctcgtgaac 2400 aatctgggca cgatcgcgcg ctccggcacg aaggcgttca tggaggcactggaggccggc 2460 ggcgacatga gcatgatcgg ccagttcggt gtcggcttct actccgcgtaccttgtggcg 2520 gaccgcgtga cggtggtgtc gaagaacaac tcggacgagg cgtacgtatgggagtcgtcc 2580 gcgggcggca cgttcaccat cacgagcgtg ccggagtcgg acatgaagcgcggcacgcgc 2640 atcacgctgc acctaaagga ggaccagcag gagtacctgg aggagcgccgggtgaaggag 2700 ctgatcaaga agcactccga gttcatcggc tacgacatcg agctgatggtggagaagacg 2760 gcggagaagg aggtgacgga cgaggacgag gaggaggacg agtcgaagaagaagtcctgc 2820 ggggacgagg gcgagccgaa ggtggaggag gtgacggagg gcggcgaggacaagaagaag 2880 aagacgaaga aggtgaagga ggtgacgaag acgtacgagg tccagaacaagcacaagccg 2940 ctctggacgc gcgacccgaa ggacgtgacg aaggaggagt acgcggccttctacaaggcc 3000 atctccaacg actgggagga cccggcggcg acgaagcact tctcggtggagggccagctg 3060 gagttccgcg cgatcgcgtt cgtgccgaag cgcgcgccgt tcgacatgttcgagccgaac 3120 aagaagcgca acaacatcaa gctgtacgtg cgccgcgtgt tcatcatggacaactgcgag 3180 gacctgtgcc cggactggct cggcttcgtg aagggcgtcg tggacagcgaggacctgccg 3240 ctgaacatct cgcgcgagaa cctgcagcag aacaagatcc tgaaggtgatccgcaagaac 3300 atcgtgaaga agtgcctgga gctgttcgaa gagatagcgg agaacaaggaggactacaag 3360 cagttctacg agcagttcgg caagaacatc aagctgggca tccacgaggacacggcgaac 3420 cgcaagaagc tgatggagtt gctgcgcttc tacagcaccg agtcgggggaggagatgacg 3480 acactgaagg actacgtgac gcgcatgaag ccggagcaga agtcgatctactacatcact 3540 ggcgacagca agaagaagct ggagtcgtcg ccgttcatcg agaaggcgagacgctgcggg 3600 ctcgaggtgc tgttcatgac ggagccgatc gacgagtacg tgatgcagcaggtgaaggac 3660 ttcgaggaca agaagttcgc gtgcctgacg aaggaaggcg tgcacttcgaggagtccgag 3720 gaggagaaga agcagcgcga ggagaagaag gcggcgtgcg agaagctgtgcaagacgatg 3780 aaggaggtgc tgggcgacaa ggtggagaag gtgaccgtgt cggagcgcctgtcgacgtcg 3840 ccgtgcatcc tggtgacgtc ggagtttggg tggtcggcgc acatggaacagatcatgcgc 3900 aaccaggcgc tgcgcgactc cagcatggcg cagtacatgg tgtccaagaagacgatggag 3960 gtgaaccccg accaccccat catcaaggag ctgcgccgcc gcgtggaggcggacgagaac 4020 gacaaggccg tgaaggacct cgtcttcctg ctcttcgaca cgtcgctgctcacgtccggc 4080 ttccagctgg atgaccccac cggctacgcc gagcgcatca accgcatgatcaagctcggc 4140 ctgtcgctcg acgaggagga ggaggaggtc gccgaggcgc cgccggccgaggcagccccc 4200 gcggaggtca ccgccggcac ctccagcatg gagcaggtgg acgatatcatggcgcagaat 4260 gataagatcg ccccccagga ccaggactcc ttcctcgatg accagcccggcgttcgcccg 4320 atcccgtcct tcgacgacat gccgctgcac cagaacctgc tgcgtggcatctactcgtac 4380 gggttcgaga agccgtccag catccagcag cgcgcgatag cccccttcacgcgcggcggc 4440 gacatcatcg cgcaggccca gtccggtacc ggcaagacgg gtgccttctccatcggtctg 4500 ctgcagcgcc tggacttccg ccacaacctg atccagggcc tcgtgctctcccccactcgc 4560 gagctggccc tgcagacggc ggaggtgatc agccgcatcg gtgagttcctgtcgaacagc 4620 tccaagttct gcgagacctt tgtcggcggc acgcgcgtgc aggatgacctgcgcaagctg 4680 caggccggcg tcatcgttgc cgtgggcacg ccgggccgcg tgtccgacgtgatcaagcgt 4740 ggcgcgctgc gcacagagtc gctgcgcgtg ctggtgctcg acgaggctgatgagatgctg 4800 tctcagggct tcgcggacca gatttacgag atcttccgct tcctgccgaaggacatccag 4860 gtcgcgctct tctccgccac gatgccggag gaggtactgg agctgacgaagaagttcatg 4920 cgcgactaa 4929 <210> SEQ ID NO 99 <211> LENGTH: 4233<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: DNA sequence encoding fusion (poly-protein)constructs comprising multiple Leishmania antigens <400> SEQUENCE: 99ccagtgtggt ggatgtcctg cggtaacgcc aagatcaact ctcccgcgcc gtccttcgag 60gaggtggcgc tcatgcccaa cggcagcttc aagaagatca gcctctcctc ctacaagggc 120aagtgggtcg tgctcttctt ctacccgctc gacttcacct tcgtgtgccc gacagaggtc 180atcgcgttct ccgacagcgt gagtcgcttc aacgagctca actgcgaggt cctcgcgtgc 240tcgatagaca gcgagtacgc gcacctgcag tggacgctgc aggaccgcaa gaagggcggc 300ctcgggacca tggcgatccc aatgctagcc gacaagacca agagcatcgc tcgttcctac 360ggcgtgctgg aggagagcca gggcgtggcc taccgcggtc tcttcatcat cgacccccat 420ggcatgctgc gtcagatcac cgtcaatgac atgccggtgg gccgcagcgt ggaggaggtt 480ctacgcctgc tggaggcttt tcagttcgtg gagaagcacg gcgaggtgtg ccccgcgaac 540tggaagaagg gcgcccccac gatgaagccg gaaccgaatg cgtctgtcga gggatacttc 600agcaagcagg gatccatgga cgcaactgag ctgaagaaca aggggaacga agagttctcc 660gccggccgct atgtggaggc ggtgaactac ttctcaaagg cgatccagtt ggatgagcag 720aacagtgtcc tctacagcaa ccgctccgcc tgttttgcag ccatgcagaa atacaaggac 780gcgctggacg acgccgacaa gtgcatctcg atcaagccga attgggccaa gggctacgtg 840cgccgaggag cagctctcca tggcatgcgc cgctacgacg atgccattgc cgcgtatgaa 900aaggggctca aggtggaccc ttccaacagc ggctgcgcgc agggcgtgaa ggacgtgcag 960gtagccaagg cccgcgaagc acgtgacccc atcgctcgcg tcttcacccc ggaggcgttc 1020cgcaagatcc aagagaatcc caagctgtct ctacttatgc tgcagccgga ctacgtgaag 1080atggtagaca ccgtcatccg cgacccttcg cagggccggc tgtacatgga agaccagcgc 1140tttgccctga cgctcatgta cctgagcgga atgaagattc ccaacgatgg tgatggcgag 1200gaggaggaac gtccgtctgc gaaggcggca gagacagcga agccaaaaga ggagaagcct 1260ctcaccgaca acgagaagga ggccctggcg ctcaaggagg agggcaacaa gctgtacctc 1320tcgaagaagt ttgaggaggc gctgaccaag taccaagagg cgcaggtgaa agaccccaac 1380aacactttat acattctgaa cgtgtcggcc gtgtacttcg agcagggtga ctacgacaag 1440tgcatcgccg agtgcgagca cggtatcgag cacggtcgcg agaaccactg cgactacaca 1500atcattgcga agctcatgac ccggaacgcc ttgtgcctcc agaggcagag gaagtacgag 1560gctgctatcg acctttacaa gcgcgccctt gtcgagtggc gtaaccctga caccctcaag 1620aagctgacgg agtgcgagaa ggagcaccaa aaggcggtgg aggaagccta catcgatcct 1680gagatcgcga agcagaagaa agacgaaggt aaccagtact tcaaggagga taagttcccc 1740gaggccgtgg cagcgtacac ggaggccatc aagcgcaacc ctgccgagca cacctcctac 1800agcaatcgcg cggccgcgta catcaagctt ggagccttca acgacgccct caaggacgcg 1860gagaagtgca ttgagctgaa gcccgacttt gttaagggct acgcgcgcaa gggtcatgct 1920tacttttgga ccaagcagta caaccgcgcg ctgcaggcgt acaatgaggg cctcaaggtg 1980gacccgagca atgcggactg caaggatggg cggtatcgca caatcatgaa gattcaggag 2040atggcatctg gccaatccgc ggatggcgac gaggcggcgc gccgggccat ggacgatcct 2100gaaatcgcgg caatcatgca agatagctac atgcaactag tgttgaagga gatgcagaac 2160gatcccacgc gcattcagga gtacatgaag gactccggga tctcatcgaa gatcaacaag 2220ctgatttcag ctggcatcat tcgttttggt caggaattca gcctgacgga cccggcggtg 2280ctgggcgagg agactcacct gcgcgtccgc gtggtgccgg acaaggcgaa caagacgctg 2340acggtggagg ataacggcat cggcatgacc aaggcggacc tcgtgaacaa tctgggcacg 2400atcgcgcgct ccggcacgaa ggcgttcatg gaggcactgg aggccggcgg cgacatgagc 2460atgatcggcc agttcggtgt cggcttctac tccgcgtacc ttgtggcgga ccgcgtgacg 2520gtggtgtcga agaacaactc ggacgaggcg tacgtatggg agtcgtccgc gggcggcacg 2580ttcaccatca cgagcgtgcc ggagtcggac atgaagcgcg gcacgcgcat cacgctgcac 2640ctaaaggagg accagcagga gtacctggag gagcgccggg tgaaggagct gatcaagaag 2700cactccgagt tcatcggcta cgacatcgag ctgatggtgg agaagacggc ggagaaggag 2760gtgacggacg aggacgagga ggaggacgag tcgaagaaga agtcctgcgg ggacgagggc 2820gagccgaagg tggaggaggt gacggagggc ggcgaggaca agaagaagaa gacgaagaag 2880gtgaaggagg tgacgaagac gtacgaggtc cagaacaagc acaagccgct ctggacgcgc 2940gacccgaagg acgtgacgaa ggaggagtac gcggccttct acaaggccat ctccaacgac 3000tgggaggacc cggcggcgac gaagcacttc tcggtggagg gccagctgga gttccgcgcg 3060atcgcgttcg tgccgaagcg cgcgccgttc gacatgttcg agccgaacaa gaagcgcaac 3120aacatcaagc tgtacgtgcg ccgcgtgttc atcatggaca actgcgagga cctgtgcccg 3180gactggctcg gcttcgtgaa gggcgtcgtg gacagcgagg acctgccgct gaacatctcg 3240cgcgagaacc tgcagcagaa caagatcctg aaggtgatcc gcaagaacat cgtgaagaag 3300tgcctggagc tgttcgaaga gatagcggag aacaaggagg actacaagca gttctacgag 3360cagttcggca agaacatcaa gctgggcatc cacgaggaca cggcgaaccg caagaagctg 3420atggagttgc tgcgcttcta cagcaccgag tcgggggagg agatgacgac actgaaggac 3480tacgtgacgc gcatgaagcc ggagcagaag tcgatctact acatcactgg cgacagcaag 3540aagaagctgg agtcgtcgcc gttcatcgag aaggcgagac gctgcgggct cgaggtgctg 3600ttcatgacgg agccgatcga cgagtacgtg atgcagcagg tgaaggactt cgaggacaag 3660aagttcgcgt gcctgacgaa ggaaggcgtg cacttcgagg agtccgagga ggagaagaag 3720cagcgcgagg agaagaaggc ggcgtgcgag aagctgtgca agacgatgaa ggaggtgctg 3780ggcgacaagg tggagaaggt gaccgtgtcg gagcgcctgt cgacgtcgcc gtgcatcctg 3840gtgacgtcgg agtttgggtg gtcggcgcac atggaacaga tcatgcgcaa ccaggcgctg 3900cgcgactcca gcatggcgca gtacatggtg tccaagaaga cgatggaggt gaaccccgac 3960caccccatca tcaaggagct gcgccgccgc gtggaggcgg acgagaacga caaggccgtg 4020aaggacctcg tcttcctgct cttcgacacg tcgctgctca cgtccggctt ccagctggat 4080gaccccaccg gctacgccga gcgcatcaac cgcatgatca agctcggcct gtcgctcgac 4140gaggaggagg aggaggtcgc cgaggcgccg ccggccgagg cagcccccgc ggaggtcacc 4200gccggcacct ccagcatgga gcaggtggac taa 4233 <210> SEQ ID NO 100 <211>LENGTH: 4917 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: DNA sequence encoding fusion(poly-protein) constructs comprising multiple Leishmania antigens <400>SEQUENCE: 100 ccagtgtggt ggatgtcctg cggtaacgcc aagatcaact ctcccgcgccgtccttcgag 60 gaggtggcgc tcatgcccaa cggcagcttc aagaagatca gcctctcctcctacaagggc 120 aagtgggtcg tgctcttctt ctacccgctc gacttcacct tcgtgtgcccgacagaggtc 180 atcgcgttct ccgacagcgt gagtcgcttc aacgagctca actgcgaggtcctcgcgtgc 240 tcgatagaca gcgagtacgc gcacctgcag tggacgctgc aggaccgcaagaagggcggc 300 ctcgggacca tggcgatccc aatgctagcc gacaagacca agagcatcgctcgttcctac 360 ggcgtgctgg aggagagcca gggcgtggcc taccgcggtc tcttcatcatcgacccccat 420 ggcatgctgc gtcagatcac cgtcaatgac atgccggtgg gccgcagcgtggaggaggtt 480 ctacgcctgc tggaggcttt tcagttcgtg gagaagcacg gcgaggtgtgccccgcgaac 540 tggaagaagg gcgcccccac gatgaagccg gaaccgaatg cgtctgtcgagggatacttc 600 agcaagcagg gatccatgga cgcaactgag ctgaagaaca aggggaacgaagagttctcc 660 gccggccgct atgtggaggc ggtgaactac ttctcaaagg cgatccagttggatgagcag 720 aacagtgtcc tctacagcaa ccgctccgcc tgttttgcag ccatgcagaaatacaaggac 780 gcgctggacg acgccgacaa gtgcatctcg atcaagccga attgggccaagggctacgtg 840 cgccgaggag cagctctcca tggcatgcgc cgctacgacg atgccattgccgcgtatgaa 900 aaggggctca aggtggaccc ttccaacagc ggctgcgcgc agggcgtgaaggacgtgcag 960 gtagccaagg cccgcgaagc acgtgacccc atcgctcgcg tcttcaccccggaggcgttc 1020 cgcaagatcc aagagaatcc caagctgtct ctacttatgc tgcagccggactacgtgaag 1080 atggtagaca ccgtcatccg cgacccttcg cagggccggc tgtacatggaagaccagcgc 1140 tttgccctga cgctcatgta cctgagcgga atgaagattc ccaacgatggtgatggcgag 1200 gaggaggaac gtccgtctgc gaaggcggca gagacagcga agccaaaagaggagaagcct 1260 ctcaccgaca acgagaagga ggccctggcg ctcaaggagg agggcaacaagctgtacctc 1320 tcgaagaagt ttgaggaggc gctgaccaag taccaagagg cgcaggtgaaagaccccaac 1380 aacactttat acattctgaa cgtgtcggcc gtgtacttcg agcagggtgactacgacaag 1440 tgcatcgccg agtgcgagca cggtatcgag cacggtcgcg agaaccactgcgactacaca 1500 atcattgcga agctcatgac ccggaacgcc ttgtgcctcc agaggcagaggaagtacgag 1560 gctgctatcg acctttacaa gcgcgccctt gtcgagtggc gtaaccctgacaccctcaag 1620 aagctgacgg agtgcgagaa ggagcaccaa aaggcggtgg aggaagcctacatcgatcct 1680 gagatcgcga agcagaagaa agacgaaggt aaccagtact tcaaggaggataagttcccc 1740 gaggccgtgg cagcgtacac ggaggccatc aagcgcaacc ctgccgagcacacctcctac 1800 agcaatcgcg cggccgcgta catcaagctt ggagccttca acgacgccctcaaggacgcg 1860 gagaagtgca ttgagctgaa gcccgacttt gttaagggct acgcgcgcaagggtcatgct 1920 tacttttgga ccaagcagta caaccgcgcg ctgcaggcgt acaatgagggcctcaaggtg 1980 gacccgagca atgcggactg caaggatggg cggtatcgca caatcatgaagattcaggag 2040 atggcatctg gccaatccgc ggatggcgac gaggcggcgc gccgggccatggacgatcct 2100 gaaatcgcgg caatcatgca agatagctac atgcaactag tgttgaaggagatgcagaac 2160 gatcccacgc gcattcagga gtacatgaag gactccggga tctcatcgaagatcaacaag 2220 ctgatttcag ctggcatcat tcgttttggt caggaattca gcctgacggacccggcggtg 2280 ctgggcgagg agactcacct gcgcgtccgc gtggtgccgg acaaggcgaacaagacgctg 2340 acggtggagg ataacggcat cggcatgacc aaggcggacc tcgtgaacaatctgggcacg 2400 atcgcgcgct ccggcacgaa ggcgttcatg gaggcactgg aggccggcggcgacatgagc 2460 atgatcggcc agttcggtgt cggcttctac tccgcgtacc ttgtggcggaccgcgtgacg 2520 gtggtgtcga agaacaactc ggacgaggcg tacgtatggg agtcgtccgcgggcggcacg 2580 ttcaccatca cgagcgtgcc ggagtcggac atgaagcgcg gcacgcgcatcacgctgcac 2640 ctaaaggagg accagcagga gtacctggag gagcgccggg tgaaggagctgatcaagaag 2700 cactccgagt tcatcggcta cgacatcgag ctgatggtgg agaagacggcggagaaggag 2760 gtgacggacg aggacgagga ggaggacgag tcgaagaaga agtcctgcggggacgagggc 2820 gagccgaagg tggaggaggt gacggagggc ggcgaggaca agaagaagaagacgaagaag 2880 gtgaaggagg tgacgaagac gtacgaggtc cagaacaagc acaagccgctctggacgcgc 2940 gacccgaagg acgtgacgaa ggaggagtac gcggccttct acaaggccatctccaacgac 3000 tgggaggacc cggcggcgac gaagcacttc tcggtggagg gccagctggagttccgcgcg 3060 atcgcgttcg tgccgaagcg cgcgccgttc gacatgttcg agccgaacaagaagcgcaac 3120 aacatcaagc tgtacgtgcg ccgcgtgttc atcatggaca actgcgaggacctgtgcccg 3180 gactggctcg gcttcgtgaa gggcgtcgtg gacagcgagg acctgccgctgaacatctcg 3240 cgcgagaacc tgcagcagaa caagatcctg aaggtgatcc gcaagaacatcgtgaagaag 3300 tgcctggagc tgttcgaaga gatagcggag aacaaggagg actacaagcagttctacgag 3360 cagttcggca agaacatcaa gctgggcatc cacgaggaca cggcgaaccgcaagaagctg 3420 atggagttgc tgcgcttcta cagcaccgag tcgggggagg agatgacgacactgaaggac 3480 tacgtgacgc gcatgaagcc ggagcagaag tcgatctact acatcactggcgacagcaag 3540 aagaagctgg agtcgtcgcc gttcatcgag aaggcgagac gctgcgggctcgaggtgctg 3600 ttcatgacgg agccgatcga cgagtacgtg atgcagcagg tgaaggacttcgaggacaag 3660 aagttcgcgt gcctgacgaa ggaaggcgtg cacttcgagg agtccgaggaggagaagaag 3720 cagcgcgagg agaagaaggc ggcgtgcgag aagctgtgca agacgatgaaggaggtgctg 3780 ggcgacaagg tggagaaggt gaccgtgtcg gagcgcctgt cgacgtcgccgtgcatcctg 3840 gtgacgtcgg agtttgggtg gtcggcgcac atggaacaga tcatgcgcaaccaggcgctg 3900 cgcgactcca gcatggcgca gtacatggtg tccaagaaga cgatggaggtgaaccccgac 3960 caccccatca tcaaggagct gcgccgccgc gtggaggcgg acgagaacgacaaggccgtg 4020 aaggacctcg tcttcctgct cttcgacacg tcgctgctca cgtccggcttccagctggat 4080 gaccccaccg gctacgccga gcgcatcaac cgcatgatca agctcggcctgtcgctcgac 4140 gaggaggagg aggaggtcgc cgaggcgccg ccggccgagg cagcccccgcggaggtcacc 4200 gccggcacct ccagcatgga gcaggtggac gatatcatgg cgcagaatgataagatcgcc 4260 ccccaggacc aggactcctt cctcgatgac cagcccggcg ttcgcccgatcccgtccttc 4320 gacgacatgc cgctgcacca gaacctgctg cgtggcatct actcgtacgggttcgagaag 4380 ccgtccagca tccagcagcg cgcgatagcc cccttcacgc gcggcggcgacatcatcgcg 4440 caggcccagt ccggtaccgg caagacgggt gccttctcca tcggtctgctgcagcgcctg 4500 gacttccgcc acaacctgat ccagggcctc gtgctctccc ccactcgcgagctggccctg 4560 cagacggcgg aggtgatcag ccgcatcggt gagttcctgt cgaacagctccaagttctgc 4620 gagacctttg tcggcggcac gcgcgtgcag gatgacctgc gcaagctgcaggccggcgtc 4680 atcgttgccg tgggcacgcc gggccgcgtg tccgacgtga tcaagcgtggcgcgctgcgc 4740 acagagtcgc tgcgcgtgct ggtgctcgac gaggctgatg agatgctgtctcagggcttc 4800 gcggaccaga tttacgagat cttccgcttc ctgccgaagg acatccaggtcgcgctcttc 4860 tccgccacga tgccggagga ggtactggag ctgacgaaga agttcatgcgcgactaa 4917 <210> SEQ ID NO 101 <211> LENGTH: 2735 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: DNA sequence encoding fusion (poly-protein) constructscomprising multiple Leishmania antigens <400> SEQUENCE: 101 catatgcaccaccaccacca ccacatgtcc tgcggtaacg ccaagatcaa ctctcccgcg 60 ccgtccttcgaggaggtggc gctcatgccc aacggcagct tcaagaagat cagcctctcc 120 tcctacaagggcaagtgggt cgtgctcttc ttctacccgc tcgacttcac cttcgtgtgc 180 ccgacagaggtcatcgcgtt ctccgacagc gtgagtcgct tcaacgagct caactgcgag 240 gtcctcgcgtgctcgataga cagcgagtac gcgcacctgc agtggacgct gcaggaccgc 300 aagaagggcggcctcgggac catggcgatc ccaatgctag ccgacaagac caagagcatc 360 gctcgttcctacggcgtgct ggaggagagc cagggcgtgg cctaccgcgg tctcttcatc 420 atcgacccccatggcatgct gcgtcagatc accgtcaatg acatgccggt gggccgcagc 480 gtggaggaggttctacgcct gctggaggct tttcagttcg tggagaagca cggcgaggtg 540 tgccccgcgaactggaagaa gggcgccccc acgatgaagc cggaaccgaa tgcgtctgtc 600 gagggatacttcagcaagca gggatccatg gacgcaactg agctgaagaa caaggggaac 660 gaagagttctccgccggccg ctatgtggag gcggtgaact acttctcaaa ggcgatccag 720 ttggatgagcagaacagtgt cctctacagc aaccgctccg cctgttttgc agccatgcag 780 aaatacaaggacgcgctgga cgacgccgac aagtgcatct cgatcaagcc gaattgggcc 840 aagggctacgtgcgccgagg agcagctctc catggcatgc gccgctacga cgatgccatt 900 gccgcgtatgaaaaggggct caaggtggac ccttccaaca gcggctgcgc gcagggcgtg 960 aaggacgtgcaggtagccaa ggcccgcgaa gcacgtgacc ccatcgctcg cgtcttcacc 1020 ccggaggcgttccgcaagat ccaagagaat cccaagctgt ctctacttat gctgcagccg 1080 gactacgtgaagatggtaga caccgtcatc cgcgaccctt cgcagggccg gctgtacatg 1140 gaagaccagcgctttgccct gacgctcatg tacctgagcg gaatgaagat tcccaacgat 1200 ggtgatggcgaggaggagga acgtccgtct gcgaaggcgg cagagacagc gaagccaaaa 1260 gaggagaagcctctcaccga caacgagaag gaggccctgg cgctcaagga ggagggcaac 1320 aagctgtacctctcgaagaa gtttgaggag gcgctgacca agtaccaaga ggcgcaggtg 1380 aaagaccccaacaacacttt atacattctg aacgtgtcgg ccgtgtactt cgagcagggt 1440 gactacgacaagtgcatcgc cgagtgcgag cacggtatcg agcacggtcg cgagaaccac 1500 tgcgactacacaatcattgc gaagctcatg acccggaacg ccttgtgcct ccagaggcag 1560 aggaagtacgaggctgctat cgacctttac aagcgcgccc ttgtcgagtg gcgtaaccct 1620 gacaccctcaagaagctgac ggagtgcgag aaggagcacc aaaaggcggt ggaggaagcc 1680 tacatcgatcctgagatcgc gaagcagaag aaagacgaag gtaaccagta cttcaaggag 1740 gataagttccccgaggccgt ggcagcgtac acggaggcca tcaagcgcaa ccctgccgag 1800 cacacctcctacagcaatcg cgcggccgcg tacatcaagc ttggagcctt caacgacgcc 1860 ctcaaggacgcggagaagtg cattgagctg aagcccgact ttgttaaggg ctacgcgcgc 1920 aagggtcatgcttacttttg gaccaagcag tacaaccgcg cgctgcaggc gtacaatgag 1980 ggcctcaaggtggacccgag caatgcggac tgcaaggatg ggcggtatcg cacaatcatg 2040 aagattcaggagatggcatc tggccaatcc gcggatggcg acgaggcggc gcgccgggcc 2100 atggacgatcctgaaatcgc ggcaatcatg caagatagct acatgcaact agtgttgaag 2160 gagatgcagaacgatcccac gcgcattcag gagtacatga aggactccgg gatctcatcg 2220 aagatcaacaagctgatttc agctggcatc attcgttttg gtcaggaatt ctgcagatat 2280 ccatcacactggcggccgct cgagcagatc cggctgctaa caaagcccga aaggaagctg 2340 agttggctgctgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg 2400 tcttgaggggttttttgctg aaaggaggaa ctatatccgg ataattcttg aagacgaaag 2460 ggcctcgtgatacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg 2520 tcaggtggcacttttcgggg aaatgtgcgc ggaaccccta tttggttatt tttctaaata 2580 cattcaaatatgtatccgct catgagacaa taacccytga taaatgcttc aataatattg 2640 aaaaaaggaagaatatgaag tatttcaaca tttcccgggt cccccttatt cccttttttt 2700 gccgccattttgcctttctg tttttggttc accca 2735 <210> SEQ ID NO 102 <211> LENGTH: 1713<212> TYPE: DNA <213> ORGANISM: Leishmania major <400> SEQUENCE: 102atggcgcagt gcgtgcgtcg gctggtgctg gcggcgacgc tcgccgctgc ggtggcgctg 60ctgctgtgca cgagcagtgc gccggtggcg cgtgctgctg ggacgaacga cttcactgcg 120gcgcagcgga cgaacacgct ggcggtgctg caggcgtttg ggcgtgcgat ccctgagctt 180ggggagaagt gggcgggcaa cgacttctgc tcatgggagt ttatcgtgtg taatgttata 240ggtgtgaacg tacggggaat cagtccgacg tatgccggca cgctgccgga gatacctgtg 300aacgtcgact acaggcacgt cgtgatcaag cagctcgact tttccgaaat ggggccgggg 360ctgagcggga cgctgccgga cagctggagc aagctggaag gactgacttc ccttacgttg 420tcgggcaaca aagtgagcgg tacgctgccc gcctcatggc acttgatgaa gcggttgaca 480tctttggtaa ttgcagactt tgacagtatc accggcagcc tgccgcctga gtggagctcg 540atgcctaatt taaacgctgt ggagctgaag cgactaaaac tgagcggtac gttgcctgcg 600gactggagct ctttgaaatc actgtcgaac gtcgttcttg aggacacgcc gatcacaggc 660ttgttgcccc cggagtgggc ctcgctggag agaatacagc agctggttct acggaaattg 720aagctgaccg gccctctccc tcctcagtgg agctcaatga agatattgca gtatcttact 780ctggatggca ctcaggtctc cggcacgctg ccgccccagt ggagcgcgat ggcatcggtg 840cgaattctta acctggaggg tactgaggtc tctggtacgc tgccgcctga gtggatatcg 900atgagcaggc tgcaaactct gaatctgcgg cgcacgaaag tatccggcac tctgccgccc 960gaatggagtt ctatgagcag cctggagtac tttcaccttt atcttactca ggtctccggc 1020acgctgccgc ccgagtggag tgggatgtcg aaggccgcat acttctggct ggaatactgc 1080gacctgtccg gcagtctgcc gcccgagtgg tcgtcgatgc caaagctgcg cggtatctca 1140ctgagcggca acaagttctg cgggtgtgtg ccggactcgt gggatcagaa ggctggtctt 1200gttgtgggca tcgaggacaa gcacaagggc agcgactgct tggctgctaa ggactgcaca 1260acgaccacca caaaaccccc caccacgaca acgaccccca ctaagccgcc tgccacaacc 1320accactgagg caccggctga acccacgacc accactgagg caccggctga acccacgacc 1380accactgagg caccggctga acccacgacc accactgagg caccggctga acccacaacc 1440accactgagg caccggctga acccacgacc actgctaccc caacaaacac gccgactcct 1500gcaccagaga cggagtgcga ggtggatggg tgtgaggtgt gcgaggggga ctccgctgcg 1560aggtgcgcga ggtgccgtga ggactacttc ctgacggacg agaggacgtg cctggtgtac 1620tgcgatggcg gtgttgctgc tgtgtcgagc ggagtggcag cagcagctgt tgtgtgcgtg 1680gctgtgctgt tcagcgtggg gctggcggcg tga 1713 <210> SEQ ID NO 103 <211>LENGTH: 2421 <212> TYPE: DNA <213> ORGANISM: Leishmania major <220>FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (1)...(2421) <223>OTHER INFORMATION: n=A,T,C or G <400> SEQUENCE: 103 tcgaattcggcacgagggca cgcacaagac gaagagttcc aaacagcaac gagtatacgc 60 cactgtcgaaaaacagacac gcagtagaga gaaggaggag gaggaggagg aggggggaga 120 gcaagaggcgggtgggggtt ggagggacag cgctgcgtgc cgccgtctga catgtccgtt 180 ttgatgcgtcttcgcagagt ggagggagga caccactggc gctgttggcg tgtangcaga 240 gcatcgctcggctcgtgccg aattcggcac gagcggcacg agccctcgct ctgcctggta 300 agctcagcagacaccgacgc ccgagcaatc ccgcccacgg acctgctgcc gccccgctct 360 gctcgtgaccctggctgcga atggcgcagt gcgtgcgtcg gctggtgctg gcggcgacgc 420 tcgccgctgcggtggcgctg ctgctgtgca cgagcagtgc gccggtggcg cgtgctgctg 480 ggacgaacgacttcactgcg gcgcagcgga cgaacacgct ggcggtgctg caggcgtttg 540 ggcgtgcgatccctgagctt ggggagaagt gggcgggcaa cgacttctgc tcatgggagt 600 ttatcgtgtgtaatgttata ggtgtgaacg tacggggaat cagtccgacg tatgccggca 660 cgctgccggagatacctgtg aacgtcgact acaggcacgt cgtgatcaag cagctcgact 720 tttccgaaatggggccgggg ctgagcggga cgctgccgga cagctggagc aagctggaag 780 gactgacttcccttacgttg tcgggcaaca aagtgagcgg tacgctgccc gcctcatggc 840 acttgatgaagcggttgaca tctttggtaa ttgcagactt tgacagtatc accggcagcc 900 tgccgcctgagtggagctcg atgcctaatt taaacgctgt ggagctgaag cgactaaaac 960 tgagcggtacgttgcctgcg gactggagct ctttgaaatc actgtcgaac gtcgttcttg 1020 aggacacgccgatcacaggc ttgttgcccc cggagtgggc ctcgctggag agaatacagc 1080 agctggttctacggaaattg aagctgaccg gccctctccc tcctcagtgg agctcaatga 1140 agatattgcagtatcttact ctggatggca ctcaggtctc cggcacgctg ccgccccagt 1200 ggagcgcgatggcatcggtg cgaattctta acctggaggg tactgaggtc tctggtacgc 1260 tgccgcctgagtggatatcg atgagcaggc tgcaaactct gaatctgcgg cgcacgaaag 1320 tatccggcactctgccgccc gaatggagtt ctatgagcag cctggagtac tttcaccttt 1380 atcttactcaggtctccggc acgctgccgc ccgagtggag tgggatgtcg aaggccgcat 1440 acttctggctggaatactgc gacctgtccg gcagtctgcc gcccgagtgg tcgtcgatgc 1500 caaagctgcgcggtatctca ctgagcggca acaagttctg cgggtgtgtg ccggactcgt 1560 gggatcagaaggctggtctt gttgtgggca tcgaggacaa gcacaagggc agcgactgct 1620 tggctgctaaggactgcaca acgaccacca caaaaccccc caccacgaca acgaccccca 1680 ctaagccgcctgccacaacc accactgagg caccggctga acccacgacc accactgagg 1740 caccggctgaacccacgacc accactgagg caccggctga acccacgacc accactgagg 1800 caccggctgaacccacaacc accactgagg caccggctga acccacgacc actgctaccc 1860 caacaaacacgccgactcct gcaccagaga cggagtgcga ggtggatggg tgtgaggtgt 1920 gcgagggggactccgctgcg aggtgcgcga ggtgccgtga ggactacttc ctgacggacg 1980 agaggacgtgcctggtgtac tgcgatggcg gtgttgctgc tgtgtcgagc ggagtggcag 2040 cagcagctgttgtgtgcgtg gctgtgctgt tcagcgtggg gctggcggcg tgaggacgct 2100 gctgctgttgcgcgcaggca gcggcccccg ctgcgtggca cacgactgtc tgcgtgcttg 2160 cgtgcagcgccgccccctgc gttggcgtgc gcgtgcgtgt ctctgtgagc atggctgcca 2220 gtggtgccctcgctcctgcc tctcggtgcc tctgcctctc tcggcgtgtt gatgctgtgg 2280 gctgtgtgtggggctctcat gcggcgctgc tgctcccgcg gtgtcgctcn tctgccccga 2340 ctctctctgctgccctcctc tctcgcatgc gggagaggga ggggtggcac gtgcgcgcgc 2400 gcmgttgcgcttgcgattgt g 2421 <210> SEQ ID NO 104 <211> LENGTH: 570 <212> TYPE: PRT<213> ORGANISM: Leishmania major <400> SEQUENCE: 104 Met Ala Gln Cys ValArg Arg Leu Val Leu Ala Ala Thr Leu Ala Ala 5 10 15 Ala Val Ala Leu LeuLeu Cys Thr Ser Ser Ala Pro Val Ala Arg Ala 20 25 30 Ala Gly Thr Asn AspPhe Thr Ala Ala Gln Arg Thr Asn Thr Leu Ala 35 40 45 Val Leu Gln Ala PheGly Arg Ala Ile Pro Glu Leu Gly Glu Lys Trp 50 55 60 Ala Gly Asn Asp PheCys Ser Trp Glu Phe Ile Val Cys Asn Val Ile 65 70 75 80 Gly Val Asn ValArg Gly Ile Ser Pro Thr Tyr Ala Gly Thr Leu Pro 85 90 95 Glu Ile Pro ValAsn Val Asp Tyr Arg His Val Val Ile Lys Gln Leu 100 105 110 Asp Phe SerGlu Met Gly Pro Gly Leu Ser Gly Thr Leu Pro Asp Ser 115 120 125 Trp SerLys Leu Glu Gly Leu Thr Ser Leu Thr Leu Ser Gly Asn Lys 130 135 140 ValSer Gly Thr Leu Pro Ala Ser Trp His Leu Met Lys Arg Leu Thr 145 150 155160 Ser Leu Val Ile Ala Asp Phe Asp Ser Ile Thr Gly Ser Leu Pro Pro 165170 175 Glu Trp Ser Ser Met Pro Asn Leu Asn Ala Val Glu Leu Lys Arg Leu180 185 190 Lys Leu Ser Gly Thr Leu Pro Ala Asp Trp Ser Ser Leu Lys SerLeu 195 200 205 Ser Asn Val Val Leu Glu Asp Thr Pro Ile Thr Gly Leu LeuPro Pro 210 215 220 Glu Trp Ala Ser Leu Glu Arg Ile Gln Gln Leu Val LeuArg Lys Leu 225 230 235 240 Lys Leu Thr Gly Pro Leu Pro Pro Gln Trp SerSer Met Lys Ile Leu 245 250 255 Gln Tyr Leu Thr Leu Asp Gly Thr Gln ValSer Gly Thr Leu Pro Pro 260 265 270 Gln Trp Ser Ala Met Ala Ser Val ArgIle Leu Asn Leu Glu Gly Thr 275 280 285 Glu Val Ser Gly Thr Leu Pro ProGlu Trp Ile Ser Met Ser Arg Leu 290 295 300 Gln Thr Leu Asn Leu Arg ArgThr Lys Val Ser Gly Thr Leu Pro Pro 305 310 315 320 Glu Trp Ser Ser MetSer Ser Leu Glu Tyr Phe His Leu Tyr Leu Thr 325 330 335 Gln Val Ser GlyThr Leu Pro Pro Glu Trp Ser Gly Met Ser Lys Ala 340 345 350 Ala Tyr PheTrp Leu Glu Tyr Cys Asp Leu Ser Gly Ser Leu Pro Pro 355 360 365 Glu TrpSer Ser Met Pro Lys Leu Arg Gly Ile Ser Leu Ser Gly Asn 370 375 380 LysPhe Cys Gly Cys Val Pro Asp Ser Trp Asp Gln Lys Ala Gly Leu 385 390 395400 Val Val Gly Ile Glu Asp Lys His Lys Gly Ser Asp Cys Leu Ala Ala 405410 415 Lys Asp Cys Thr Thr Thr Thr Thr Lys Pro Pro Thr Thr Thr Thr Thr420 425 430 Pro Thr Lys Pro Pro Ala Thr Thr Thr Thr Glu Ala Pro Ala GluPro 435 440 445 Thr Thr Thr Thr Glu Ala Pro Ala Glu Pro Thr Thr Thr ThrGlu Ala 450 455 460 Pro Ala Glu Pro Thr Thr Thr Thr Glu Ala Pro Ala GluPro Thr Thr 465 470 475 480 Thr Thr Glu Ala Pro Ala Glu Pro Thr Thr ThrAla Thr Pro Thr Asn 485 490 495 Thr Pro Thr Pro Ala Pro Glu Thr Glu CysGlu Val Asp Gly Cys Glu 500 505 510 Val Cys Glu Gly Asp Ser Ala Ala ArgCys Ala Arg Cys Arg Glu Asp 515 520 525 Tyr Phe Leu Thr Asp Glu Arg ThrCys Leu Val Tyr Cys Asp Gly Gly 530 535 540 Val Ala Ala Val Ser Ser GlyVal Ala Ala Ala Ala Val Val Cys Val 545 550 555 560 Ala Val Leu Phe SerVal Gly Leu Ala Ala 565 570 <210> SEQ ID NO 105 <211> LENGTH: 1688 <212>TYPE: DNA <213> ORGANISM: Leishmania major <400> SEQUENCE: 105taacgctata taagtatcag tttctgtact ttattgctca tcactgccgt ttgactgccg 60cgggcattgg cgctaccact ttcctcactc tttatccctt cagcattgtt tcgtacacac 120gcacgcgcac gtgaaagagc cgcacgccga cagagcagcc gttccggact ctccgataac 180tgaacgccac ccacccaaaa aaaatgtcaa agaacgctga ccaggaggag tgggaggatt 240acggcgacga ggaggtgcag gatgaagaag aggaggacac caccatcaac aactccgacg 300tggtggtgcg ctacaagaag gccgcaacgt ggtgcaatga aacgttgcgc gtgcttatcg 360atgccacaaa acctggcgcc aaggtgtgcg acctgtgccg cctcggtgat gacaccatca 420ccgccaaggt caagacaatg ttcaaaggca cggaaaaagg catcgctttc ccgacctgca 480tctcggtcaa caactgcgta tgccacaaca gccctggcgt gtcggacgag acgacgcagc 540aagagatcgc gatgggtgac gtcgtgcact acgacctggg catccacgtg gacggctact 600gcgccgtcgt cgcgcacacc attcaggtga cagaggacaa tgagcttggc aaggacgaga 660aggcggcgcg cgtcattaca gcggcgtaca acatcctgaa cacggcgctg cgccagatgc 720gtcccggtac gaccatctac caggtgacag acgtagttga gaaggctgcg gagcactaca 780aggtgactcc ggtagacggc gtcctctcgc atatgatgaa gcgctacatc atagacggat 840accgctgtat cccgcagcgc agggtcgcgg agcacatggt gcacgactac gatctcgaga 900aagcgcaggt gtggacgcta gacattgtca tgacctccgg caagggcaag ctgaaggagc 960gcgatgcgcg gccgtgcgtg ttcaaggtgg ctctggactc caactactct gtgaaaatgg 1020aaagcgcgaa ggaggttcag aaggaaatcg actccaagta tgccaccttc ccctttgcca 1080tccgcaacct ggaggccaag aaggcccgcc tcggtctcaa cgagatggcg aagcacggtg 1140ctgtcatccc gtaccctatt ctcttcgaaa aggaaggcga ggtcgtcgcc catttcaaga 1200ttacggtgct catcagcaac aagaagattg agccgattac cggcctgaag ccgcagaagg 1260ccccggcgct cgagccatac acggacgaga tgctgcttgc gacgaacaag ctctcgctgt 1320cgctagagaa gaaggcggcg aagtagacgg ccgtggcatc cgtgacgctg tactgcgagc 1380tttcgtaggc gtacgcctct tgtgaggcgt acacgtgtgc tgtttgcgga cgaggaggca 1440cccattctgt tccccttctt cgctaatctc cgcgtttcct ctgacgctgg cttctctgcc 1500ggagtgtggt gaggcgcgtg ggggagaaac ggcccactcg catgcctgtg catacgcgag 1560cacggtaggg agcgcggtgt gtgtgtgtgt gggggggcgt gttacgagta caaaagaggc 1620tcgatctctg cgactctttt ctttctgtaa acagggaaca taagtaacca aaaaaaaaaa 1680aaaaaaaa 1688 210> SEQ ID NO 106 <211> LENGTH: 380 <212> TYPE: PRT <213>ORGANISM: Leishmania major <400> SEQUENCE: 106 Met Ser Lys Asn Ala AspGln Glu Glu Trp Glu Asp Tyr Gly Asp Glu 1 5 10 15 Glu Val Gln Asp GluGlu Glu Glu Asp Thr Thr Ile Asn Asn Ser Asp 20 25 30 Val Val Val Arg TyrLys Lys Ala Ala Thr Trp Cys Asn Glu Thr Leu 35 40 45 Arg Val Leu Ile AspAla Thr Lys Pro Gly Ala Lys Val Cys Asp Leu 50 55 60 Cys Arg Leu Gly AspAsp Thr Ile Thr Ala Lys Val Lys Thr Met Phe 65 70 75 80 Lys Gly Thr GluLys Gly Ile Ala Phe Pro Thr Cys Ile Ser Val Asn 85 90 95 Asn Cys Val CysHis Asn Ser Pro Gly Val Ser Asp Glu Thr Thr Gln 100 105 110 Gln Glu IleAla Met Gly Asp Val Val His Tyr Asp Leu Gly Ile His 115 120 125 Val AspGly Tyr Cys Ala Val Val Ala His Thr Ile Gln Val Thr Glu 130 135 140 AspAsn Glu Leu Gly Lys Asp Glu Lys Ala Ala Arg Val Ile Thr Ala 145 150 155160 Ala Tyr Asn Ile Leu Asn Thr Ala Leu Arg Gln Met Arg Pro Gly Thr 165170 175 Thr Ile Tyr Gln Val Thr Asp Val Val Glu Lys Ala Ala Glu His Tyr180 185 190 Lys Val Thr Pro Val Asp Gly Val Leu Ser His Met Met Lys ArgTyr 195 200 205 Ile Ile Asp Gly Tyr Arg Cys Ile Pro Gln Arg Arg Val AlaGlu His 210 215 220 Met Val His Asp Tyr Asp Leu Glu Lys Ala Gln Val TrpThr Leu Asp 225 230 235 240 Ile Val Met Thr Ser Gly Lys Gly Lys Leu LysGlu Arg Asp Ala Arg 245 250 255 Pro Cys Val Phe Lys Val Ala Leu Asp SerAsn Tyr Ser Val Lys Met 260 265 270 Glu Ser Ala Lys Glu Val Gln Lys GluIle Asp Ser Lys Tyr Ala Thr 275 280 285 Phe Pro Phe Ala Ile Arg Asn LeuGlu Ala Lys Lys Ala Arg Leu Gly 290 295 300 Leu Asn Glu Met Ala Lys HisGly Ala Val Ile Pro Tyr Pro Ile Leu 305 310 315 320 Phe Glu Lys Glu GlyGlu Val Val Ala His Phe Lys Ile Thr Val Leu 325 330 335 Ile Ser Asn LysLys Ile Glu Pro Ile Thr Gly Leu Lys Pro Gln Lys 340 345 350 Ala Pro AlaLeu Glu Pro Tyr Thr Asp Glu Met Leu Leu Ala Thr Asn 355 360 365 Lys LeuSer Leu Ser Leu Glu Lys Lys Ala Ala Lys 370 375 380 <210> SEQ ID NO 107<211> LENGTH: 1565 <212> TYPE: DNA <213> ORGANISM: Leishmania major<400> SEQUENCE: 107 taacgctata taagtatcag tttctgtact ttattgctcttcgctctcgt tcttcgaaca 60 aacaccttta aaccgccttc caacccctct ttcttctttttcagccatgc gtgaggctat 120 ctgcatccac atcggccagg ccggctgcca ggtcggtaacgcgtgctggg agctgttctg 180 ccttgagcac ggcatccagc ctgatggctc catgccctctgacaagtgca tcggtgttga 240 ggatgacgcg ttcaacacgt tcttctcgga gaccggtgctggcaagcacg ttccgcgctg 300 catcttcctg gacctcgagc ctacggtcgt ggatgaggtgcgcaccggca cgtaccgcca 360 gctgttcaac cccgagcagc tggtgtctgg caaggaggatgcggcgaaca actacgctcg 420 tggccactac acgatcggca aggagatcgt cgaccttgcgctggaccgca ttcgcaagct 480 ggcggacaac tgcactggtc tccagggctt tatggtgttccacgctgtgg gtggcggcac 540 cggctctggc ctcggtgcgc tgctgctgga gcgcctgtctgtggactacg gcaagaagtc 600 caagctcggc tacaccgtgt acccgagccc gcaggtgtcgactgccgtcg tggagccgta 660 caactgcgtg ctgtcgacgc actcgctgct cgagcacaccgatgttgcga cgatgctcga 720 caatgaggcc atctacgacc tcactcgtcg ttctctcgacattgagcgcc cgtcgtacac 780 gaacgtgaac cgcctgatcg gccaggtggt gtcgtctctgacggcgtcgc tgcgcttcga 840 tggtgcgctg aacgtggacc tgacggagtt ccagacgaaccttgtgccgt acccgcgcat 900 ccacttcgtg ctgacgagct acgctccggt ggtgtctgccgagaaggcgt accacgagca 960 gctgtccgtc gcggacatca cgaactcggt gtttgagcctgctggcatgc tgacgaagtg 1020 cgatcctcgc cacggcaagt acatgtcgtg ctgcctcatgtaccgcggtg atgtcgtgcc 1080 gaaggatgtc aacgccgcga ttgcgacgat caagacgaagcgcacaattc agttcgtgga 1140 ctggtgcccg accggcttca agtgcggcat caactaccagccgccgaccg ttgtgcccgg 1200 cggtgacctc gcgaaggtgc agcgcgccgt gtgcatgattgccaactcga ccgcgatcgc 1260 tgaggtgttt gcccgcatcg accacaagtt cgacctgatgtacagcaagc gcgcgtttgt 1320 gcactggtac gtgggtgagg gcatggagga gggcgagttctccgaggcgc gcgaggatct 1380 cgctgcgctg gagaaggact acgaggaggt tggcgccgagtccgccgacg acatgggcga 1440 ggaggacgtc gaggagtact aaggtagact cgtgccgcgcgctgatgatg taggtgcacg 1500 cgtgcgtgtg ctgcagcgga gccgccgcca ccgcgactgtgtgtgtgtgc gcgcgtgacg 1560 accgg 1565 <210> SEQ ID NO 108 <211> LENGTH:451 <212> TYPE: PRT <213> ORGANISM: Leishmania major <400> SEQUENCE: 108Met Arg Glu Ala Ile Cys Ile His Ile Gly Gln Ala Gly Cys Gln Val 1 5 1015 Gly Asn Ala Cys Trp Glu Leu Phe Cys Leu Glu His Gly Ile Gln Pro 20 2530 Asp Gly Ser Met Pro Ser Asp Lys Cys Ile Gly Val Glu Asp Asp Ala 35 4045 Phe Asn Thr Phe Phe Ser Glu Thr Gly Ala Gly Lys His Val Pro Arg 50 5560 Cys Ile Phe Leu Asp Leu Glu Pro Thr Val Val Asp Glu Val Arg Thr 65 7075 80 Gly Thr Tyr Arg Gln Leu Phe Asn Pro Glu Gln Leu Val Ser Gly Lys 8590 95 Glu Asp Ala Ala Asn Asn Tyr Ala Arg Gly His Tyr Thr Ile Gly Lys100 105 110 Glu Ile Val Asp Leu Ala Leu Asp Arg Ile Arg Lys Leu Ala AspAsn 115 120 125 Cys Thr Gly Leu Gln Gly Phe Met Val Phe His Ala Val GlyGly Gly 130 135 140 Thr Gly Ser Gly Leu Gly Ala Leu Leu Leu Glu Arg LeuSer Val Asp 145 150 155 160 Tyr Gly Lys Lys Ser Lys Leu Gly Tyr Thr ValTyr Pro Ser Pro Gln 165 170 175 Val Ser Thr Ala Val Val Glu Pro Tyr AsnCys Val Leu Ser Thr His 180 185 190 Ser Leu Leu Glu His Thr Asp Val AlaThr Met Leu Asp Asn Glu Ala 195 200 205 Ile Tyr Asp Leu Thr Arg Arg SerLeu Asp Ile Glu Arg Pro Ser Tyr 210 215 220 Thr Asn Val Asn Arg Leu IleGly Gln Val Val Ser Ser Leu Thr Ala 225 230 235 240 Ser Leu Arg Phe AspGly Ala Leu Asn Val Asp Leu Thr Glu Phe Gln 245 250 255 Thr Asn Leu ValPro Tyr Pro Arg Ile His Phe Val Leu Thr Ser Tyr 260 265 270 Ala Pro ValVal Ser Ala Glu Lys Ala Tyr His Glu Gln Leu Ser Val 275 280 285 Ala AspIle Thr Asn Ser Val Phe Glu Pro Ala Gly Met Leu Thr Lys 290 295 300 CysAsp Pro Arg His Gly Lys Tyr Met Ser Cys Cys Leu Met Tyr Arg 305 310 315320 Gly Asp Val Val Pro Lys Asp Val Asn Ala Ala Ile Ala Thr Ile Lys 325330 335 Thr Lys Arg Thr Ile Gln Phe Val Asp Trp Cys Pro Thr Gly Phe Lys340 345 350 Cys Gly Ile Asn Tyr Gln Pro Pro Thr Val Val Pro Gly Gly AspLeu 355 360 365 Ala Lys Val Gln Arg Ala Val Cys Met Ile Ala Asn Ser ThrAla Ile 370 375 380 Ala Glu Val Phe Ala Arg Ile Asp His Lys Phe Asp LeuMet Tyr Ser 385 390 395 400 Lys Arg Ala Phe Val His Trp Tyr Val Gly GluGly Met Glu Glu Gly 405 410 415 Glu Phe Ser Glu Ala Arg Glu Asp Leu AlaAla Leu Glu Lys Asp Tyr 420 425 430 Glu Glu Val Gly Ala Glu Ser Ala AspAsp Met Gly Glu Glu Asp Val 435 440 445 Glu Glu Tyr 450 <210> SEQ ID NO109 <211> LENGTH: 1908 <212> TYPE: DNA <213> ORGANISM: Leishmania major<220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (1)...(1908)<223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 109 taacgctatataagtatcag tttctgtact ttattgtaag cgcaatcgag tttcaacagc 60 taacaaaatggtgaacttta ccgtcgatca ggtccgcgag ctgatggact atccggacca 120 gatccggaacatgtccgtga ttgctcacgt cgaccacggc aagtcgacgc tgtccgactc 180 tctcgttggtgctgccggca tcatcaagat ggaggaggct ggcgataagc ggatcatgga 240 tacacgcgcggatgagatcg cgcgtggtat cacgatcaag tccaccgcca tctccatgca 300 ctaccacgtgccgaaggaga tgatcggcga tctggatgac gacaagcgcg acttcctgat 360 caacctgatcgactcccccg gacacgtcga cttcagctcc gaggtgactg ccgctcttcg 420 tgtgacggacggcgcgctgg tcgtggtgga ctgcgtggag ggcgtgtgcg tgcagacgga 480 gacggtgctgcgccaggcgc tgacggagcg catccgccct gttgtgttca tcaacaaggt 540 ggaccgcgccatccttgagc tccaactgga ccccgaggag gcgtaccagg gcttcgtgaa 600 gacgctgcagaacgtgaacg tggtggttgc cacgtacaat gaccccagca tgggcgacgt 660 gcaggtgtcccccgagaagg gcactgtggc gatcggctct ggcctgcagg cgtgggcgtt 720 ctcgctgacccgctttgcga acatgtatgc ggcgaagttc ggcgtggacg agctgaagat 780 gcgcgagcgcctgtggggcg acaacttctt tgacgcgaag aacaagaagt ggatcaagca 840 ggagacgaacgccgatggcg agcgcgtgcg ccgcgcgttc tgccagttct gcctagaccc 900 catctaccagatcttcgacg ctgtgatgaa cgagaagaag gacaaggtgg acaagatgct 960 caagtcgctgcacgtgacgc tgacggctga ggagcgcgag caggtgccga agaagcttct 1020 gaagacggtgatgatgaagt tcctgccggc tgctgagacg ctgctacaga tgatcgtggc 1080 gcacctgccgtcgcccaaga aggcgcaggc gtaccgtgcg gagatgctgt actctggcga 1140 ggcgtcgccggaggacaagt acttcatggg tatcaagaac tgcgaccccg ctgcgccgct 1200 catgctgtacatcagcaaga tggtgccgac ggccgaccgc ggccgcttct tcgcctttgg 1260 ccgcatcttctccggtaagg tgcgcagcgg ccagaaggtg cgcatcatgg gtaacaacta 1320 cgtctacggcaagaagcagg acctgtacga ggacaagcct gtgcagcgct ccgtgctgat 1380 gatgggccgctaccaggagg ccgtggagga catgccgtgc ggtaacgtgg tgggccttgt 1440 gggcgtggacaagtacatcg tgaagtccgc gacgatcacg gacgatggcg agagcccgca 1500 cccgctgcgcgacatgaagt actctgtgtc gcccgtcgtg cgtgtggccg tggaggcgaa 1560 gaacccgtccgacctgccga agcttgtgga gggcctgaag cgccttgcca agtccgaccc 1620 gctggtggtgtgcagcattg aggagtctgg cgagcacatt gttgccggcg ctggcgagct 1680 tcaccttgagatttgcctga aggatctcca ggaggacttc atgaacggcg cgccgctnaa 1740 gatctccgagccggtggtgt cgttccgcga gacggtgacg gatgtgtcgt cgcagcagtg 1800 cctgtcgaagtctgcgaaca agcacaaccg tctcttctgc cgcggtgcgc cgctnacaga 1860 gganctggcgctggcgatng angaaggcac cgctggtccc gangcgga 1908 <210> SEQ ID NO 110 <211>LENGTH: 845 <212> TYPE: PRT <213> ORGANISM: Leishmania major <220>FEATURE: <221> NAME/KEY: VARIANT <222> LOCATION: (1)...(845) <223> OTHERINFORMATION: Xaa = Any Amino Acid <400> SEQUENCE: 110 Met Val Asn PheThr Val Asp Gln Val Arg Glu Leu Met Asp Tyr Pro 1 5 10 15 Asp Gln IleArg Asn Met Ser Val Ile Ala His Val Asp His Gly Lys 20 25 30 Ser Thr LeuSer Asp Ser Leu Val Gly Ala Ala Gly Ile Ile Lys Met 35 40 45 Glu Glu AlaGly Asp Lys Arg Ile Met Asp Thr Arg Ala Asp Glu Ile 50 55 60 Ala Arg GlyIle Thr Ile Lys Ser Thr Ala Ile Ser Met His Tyr His 65 70 75 80 Val ProLys Glu Met Ile Gly Asp Leu Asp Asp Asp Lys Arg Asp Phe 85 90 95 Leu IleAsn Leu Ile Asp Ser Pro Gly His Val Asp Phe Ser Ser Glu 100 105 110 ValThr Ala Ala Leu Arg Val Thr Asp Gly Ala Leu Val Val Val Asp 115 120 125Cys Val Glu Gly Val Cys Val Gln Thr Glu Thr Val Leu Arg Gln Ala 130 135140 Leu Thr Glu Arg Ile Arg Pro Val Val Phe Ile Asn Lys Val Asp Arg 145150 155 160 Ala Ile Leu Glu Leu Gln Leu Asp Pro Glu Glu Ala Tyr Gln GlyPhe 165 170 175 Val Lys Thr Leu Gln Asn Val Asn Val Val Val Ala Thr TyrAsn Asp 180 185 190 Pro Ser Met Gly Asp Val Gln Val Ser Pro Glu Lys GlyThr Val Ala 195 200 205 Ile Gly Ser Gly Leu Gln Ala Trp Ala Phe Ser LeuThr Arg Phe Ala 210 215 220 Asn Met Tyr Ala Ala Lys Phe Gly Val Asp GluLeu Lys Met Arg Glu 225 230 235 240 Arg Leu Trp Gly Asp Asn Phe Phe AspAla Lys Asn Lys Lys Trp Ile 245 250 255 Lys Gln Glu Thr Asn Ala Asp GlyGlu Arg Val Arg Arg Ala Phe Cys 260 265 270 Gln Phe Cys Leu Asp Pro IleTyr Gln Ile Phe Asp Ala Val Met Asn 275 280 285 Glu Lys Lys Asp Lys ValAsp Lys Met Leu Lys Ser Leu His Val Thr 290 295 300 Leu Thr Ala Glu GluArg Glu Gln Val Pro Xaa Lys Leu Leu Lys Thr 305 310 315 320 Val Met MetXaa Phe Leu Pro Ala Ala Glu Thr Leu Leu Gln Met Ile 325 330 335 Val AlaHis Leu Pro Ser Pro Lys Lys Ala Gln Ala Tyr Arg Ala Glu 340 345 350 MetLeu Tyr Ser Gly Glu Ala Ser Pro Glu Asp Lys Tyr Phe Met Gly 355 360 365Ile Lys Asn Cys Asp Pro Ala Ala Pro Leu Met Leu Tyr Ile Ser Lys 370 375380 Met Val Pro Thr Ala Asp Arg Gly Arg Phe Phe Ala Phe Gly Arg Ile 385390 395 400 Phe Ser Gly Lys Val Arg Ser Gly Gln Lys Val Arg Ile Met GlyAsn 405 410 415 Asn Tyr Val Tyr Gly Lys Lys Gln Asp Leu Tyr Glu Asp LysPro Val 420 425 430 Gln Arg Ser Val Leu Met Met Gly Arg Tyr Gln Glu AlaVal Glu Asp 435 440 445 Met Pro Cys Gly Asn Val Val Gly Leu Val Gly ValAsp Lys Tyr Ile 450 455 460 Val Lys Ser Ala Thr Ile Thr Asp Asp Gly GluSer Pro His Pro Leu 465 470 475 480 Arg Asp Met Lys Tyr Ser Val Ser ProVal Val Arg Val Ala Val Glu 485 490 495 Ala Lys Asn Pro Ser Asp Leu ProLys Leu Val Glu Gly Leu Lys Arg 500 505 510 Leu Ala Lys Ser Asp Pro LeuVal Val Cys Ser Ile Glu Glu Ser Gly 515 520 525 Glu His Ile Val Ala GlyAla Gly Glu Leu His Leu Glu Ile Cys Leu 530 535 540 Lys Asp Leu Gln GluAsp Phe Met Asn Gly Ala Pro Leu Lys Ile Ser 545 550 555 560 Glu Pro ValVal Ser Phe Arg Glu Thr Val Thr Asp Val Ser Ser Gln 565 570 575 Gln CysLeu Ser Lys Ser Ala Asn Lys His Asn Arg Leu Phe Cys Arg 580 585 590 GlyAla Pro Leu Thr Glu Glu Leu Ala Leu Ala Met Glu Glu Gly Thr 595 600 605Ala Gly Pro Glu Ala Asp Pro Lys Val Arg Ala Arg Phe Leu Ala Asp 610 615620 Asn Tyr Glu Trp Asp Val Gln Glu Ala Arg Lys Ile Trp Cys Tyr Gly 625630 635 640 Pro Asp Asn Arg Gly Pro Asn Val Val Val Asp Val Thr Lys GlyVal 645 650 655 Gln Asn Met Ala Glu Met Lys Asp Ser Phe Val Ala Ala TrpGln Trp 660 665 670 Ala Thr Arg Glu Gly Val Leu Cys Asp Glu Asn Met ArgGly Val Arg 675 680 685 Val Asn Val Glu Asp Val Thr Met His Ala Asp AlaIle His Arg Gly 690 695 700 Gly Val Gln Ile Ile Pro Thr Ala Arg Arg ValPhe Tyr Ala Cys Cys 705 710 715 720 Leu Thr Ala Ser Pro Arg Leu Met GluPro Met Phe Val Val Asp Ile 725 730 735 Gln Thr Val Glu His Ala Met GlyGly Ile Tyr Gly Val Leu Thr Arg 740 745 750 Arg Arg Gly Val Ile Ile GlyGlu Glu Asn Arg Pro Gly Thr Pro Ile 755 760 765 Tyr Asn Val Arg Ala TyrLeu Pro Val Ala Glu Ser Phe Gly Phe Thr 770 775 780 Ala Asp Leu Arg AlaGly Thr Gly Gly Gln Ala Phe Pro Gln Cys Val 785 790 795 800 Phe Asp HisTrp Gln Glu Tyr Pro Gly Asp Pro Leu Glu Pro Lys Ser 805 810 815 Leu AlaAsn Thr Thr Thr Leu Gly Ile Arg Thr Arg Lys Gly Leu Lys 820 825 830 ProAsp Ile Pro Gly Leu Asp Gln Phe Met Asp Lys Leu 835 840 845 <210> SEQ IDNO 111 <211> LENGTH: 997 <212> TYPE: DNA <213> ORGANISM: Leishmaniamajor <400> SEQUENCE: 111 ggatccgccg ccaccatggt gaacgtgtgc gttgttggtgctgccggcgg cattggccag 60 tcgctgtcgc tgctgttggt gcgccagctg ccgtacgggagcacgttgtc gctgttcgac 120 gttgtgggcg ctgcaggcgt cgcagcagac ctgtcgcatgtggacaacgc cggtgtgcag 180 gtgaagtttg cggagggcaa gatcggccat aagcgcgaccctgcgctggc agagcttgcg 240 aagggcgtgg atgtgtttgt aatggtggct ggcgttccacgcaagccggg catgacgcgc 300 gacgaccttt tcaaaatcaa cgccggaatc atcctggaccttgtgctgac gtgcgcgtcg 360 tcgagtccaa aggcggtgtt ctgcattgtg acgaaccctgtgaacagcac ggtcgcgatc 420 gcggcagagg cgctgaagag ccttggcgta tacgaccgaaaccggctgct tggcgtgtcg 480 ctgctggacg ggctgcgcgc gacgtgcttc atcaacgaggcgcgcaagcc cttagtcgtg 540 tcgcaggtac cagttgttgg cgggcacagc gacacaacgattgtgccgtt gttctaccag 600 ctaccggggc cgttgccgga gcaggcgacg ctggacaagatcgtgaagcg cgtgcaggtc 660 gcaggcacag aagtggtgaa ggcgaaggcc gggcgcgggtctgcgacgct gtcgatggcg 720 gaggctggcg cgcggttcgc gttgaaggtt gtggagggtctgaccggcac gggtaacccg 780 ctggtgtacg catatgtaga cacagacggg cagcacgagacgacgttcct cgcgatccct 840 gtggtgcttg gcatgaatgg aatcgagaag cgcctgccgattggtccgct gcactcgacg 900 gaggaaacgc tgctgaaggc ggcactgccg gtgatcaagaagaatatcgt gaagggcagc 960 gagttcgcgc gctcacacct gtagcacctc agaattc 997<210> SEQ ID NO 112 <211> LENGTH: 322 <212> TYPE: PRT <213> ORGANISM:Leishmania major <400> SEQUENCE: 112 Met Val Asn Val Cys Val Val Gly AlaAla Gly Gly Ile Gly Gln Ser 1 5 10 15 Leu Ser Leu Leu Leu Val Arg GlnLeu Pro Tyr Gly Ser Thr Leu Ser 20 25 30 Leu Phe Asp Val Val Gly Ala AlaGly Val Ala Ala Asp Leu Ser His 35 40 45 Val Asp Asn Ala Gly Val Gln ValLys Phe Ala Glu Gly Lys Ile Gly 50 55 60 His Lys Arg Asp Pro Ala Leu AlaGlu Leu Ala Lys Gly Val Asp Val 65 70 75 80 Phe Val Met Val Ala Gly ValPro Arg Lys Pro Gly Met Thr Arg Asp 85 90 95 Asp Leu Phe Lys Ile Asn AlaGly Ile Ile Leu Asp Leu Val Leu Thr 100 105 110 Cys Ala Ser Ser Ser ProLys Ala Val Phe Cys Ile Val Thr Asn Pro 115 120 125 Val Asn Ser Thr ValAla Ile Ala Ala Glu Ala Leu Lys Ser Leu Gly 130 135 140 Val Tyr Asp ArgAsn Arg Leu Leu Gly Val Ser Leu Leu Asp Gly Leu 145 150 155 160 Arg AlaThr Cys Phe Ile Asn Glu Ala Arg Lys Pro Leu Val Val Ser 165 170 175 GlnVal Pro Val Val Gly Gly His Ser Asp Thr Thr Ile Val Pro Leu 180 185 190Phe Tyr Gln Leu Pro Gly Pro Leu Pro Glu Gln Ala Thr Leu Asp Lys 195 200205 Ile Val Lys Arg Val Gln Val Ala Gly Thr Glu Val Val Lys Ala Lys 210215 220 Ala Gly Arg Gly Ser Ala Thr Leu Ser Met Ala Glu Ala Gly Ala Arg225 230 235 240 Phe Ala Leu Lys Val Val Glu Gly Leu Thr Gly Thr Gly AsnPro Leu 245 250 255 Val Tyr Ala Tyr Val Asp Thr Asp Gly Gln His Glu ThrThr Phe Leu 260 265 270 Ala Ile Pro Val Val Leu Gly Met Asn Gly Ile GluLys Arg Leu Pro 275 280 285 Ile Gly Pro Leu His Ser Thr Glu Glu Thr LeuLeu Lys Ala Ala Leu 290 295 300 Pro Val Ile Lys Lys Asn Ile Val Lys GlySer Glu Phe Ala Arg Ser 305 310 315 320 His Leu <210> SEQ ID NO 113<211> LENGTH: 1617 <212> TYPE: DNA <213> ORGANISM: Leishmania major andchagasi <400> SEQUENCE: 113 cggaatacgt acctcctccc ccttcttggt agaagaacaacaacaacgtt caagacgacg 60 ccgcgccttc ttgtaccgca tttgcttctg agcacgttcaatccgtgcct tgcaaacatg 120 gaggcgtaca agaagctgga aacgatcttt acgaaggtctaccgcctgga ccacttcctc 180 ggtctgggca actgggacat gaacacaaac atgccccccaagggcgagga atcacgcggt 240 gaggcgatgg cgatgctctc ggagctccgc tttggcttcatcacggcacc ggaggtgaaa 300 agcctgattg agagtgccac caagggcagc gaggagctgaatgcggtgca gcgcgctaac 360 ttgcgggaga tgaggcgtgc gtggaagagc gccaccgccttgccggctga gtttgtgggc 420 cgcaagatgc gcctcacgac acacgcgcac agcgtgtggcgcgacagccg caaagcaaat 480 gacttcgcca agttcctacc ggtgctcagg gacctggtggcgctcgcccg tgaggagggc 540 tcatacctcg ccgccggcac ctccctctcc ccgtatgaggcgctcatgaa cgagtacgag 600 ccaggaatca cgacacaaaa gctggatgag gtgtacgcaaatgtaaagtc gtggctgccg 660 cagctgctaa aggacattgt gcagaagcag tccggcgagtcggtgattgc gttctcgcat 720 aagttcccgc aggacaagca ggaagcactg tgcaaggaattcatgaagat ctggcacttc 780 gacaccgatg ccggtcgcct cgacgtcagc ccccaccctttcacgggaat gacgaaggag 840 gactgccgac tcacaacaaa ctacatcgaa gacacgtttgttcagagctt gtatggcgtc 900 atccacgaga gtgggcatgg caagtacgag cagaactgtggcccacgcga gcacatcacg 960 cagccggtgt gcaacgcccg ctctcttggc ctgcatgagagccagagcct ctttgcggag 1020 tttcagatcg gccacgcgac gcccttcatc gactacctcacaactcgcct tcctgagttc 1080 ttcgaggcgc agccagcgtt ctcgcaggac aacatgcgcaagtcgctgca gcaggtgaag 1140 ccgggctaca ttcgcgtcga tgccgatgag gtgtgctaccctctgcacgt gatcctgcgc 1200 tacgagatcg agcgcgactt gatggagggc aaaatggaggtggaagacgt gccgcgcgcg 1260 tggaacgcaa agatgcagga gtacttgggt ctctcaacggagggccgtga cgacgttggg 1320 tgcctgcagg acgtgcattg gtccatgggt gcgctcggctactttccgac gtactcgctc 1380 ggcgccatgt atgcggcgca gatcatggcg agcatccgaaaggagctggg agacgacaag 1440 gtggatgagt gcctgcgcac cggtgagctc ggccccctcctggaaaagca gcaggagaag 1500 atctgggatc atgggtgcct gtacgagacg gacgacctcatgacgcgtgc gacgggcgag 1560 acgctgaacc ccgagtacct gcgccgccac ctggaggcgcgctacctaaa cgcctga 1617 <210> SEQ ID NO 114 <211> LENGTH: 582 <212>TYPE: DNA <213> ORGANISM: Leishmania major and chagasi <400> SEQUENCE:114 atgcatcacc atcaccatca cgatcccccg ggctgcagga attcggcacg agagctgcag 60cagcgcctag acaccgccac gcagcagcgc gccgagctgg aggcacgggt ggcacggctg 120gccgcggacc gcgacgaggc gcgccagcag ctggccgcga acgccgagga gctgcagcag 180cgcctagaca ccgccacgca gcagcgcgcc gagctggagg cacgggtggc acggctggcc 240gcggacggcg acgaggcccg ccagcagctg gccgcgaacg ccgaggagct gcagcagcgc 300ctagacaccg ccacgcagca gcgcgccgag ctggaggcac aggtggcacg gctggccgcg 360aacgccgagg agctgcagca gcgcctagac accgccacgc agcagcgcgc cgagctggag 420gcacgggtgg cacggctggc cgcggaccgc gacgaggcgc gccagcagct ggccgcgaac 480gccgaggagc tgcagcagcg cctagacacc gccacgcagc agcgcgccga gctggaggca 540caggtggcac ggctggccgc gaacgccgct cgtgccgaat aa 582 <210> SEQ ID NO 115<211> LENGTH: 7065 <212> TYPE: DNA <213> ORGANISM: Leishmania major andchagasi <400> SEQUENCE: 115 atgtccactc ctgtcagtgg agtagtcccg caggaccgctggcagcctca gcagagggtg 60 aaggtctgtc agtaccagga ctgcggcgcc cccttcggcttcttctccac caaggtcaac 120 tgccaccgct gcggcatcgt cctctgcagc aagtgcgccgccaccaagac cgtgattccc 180 cgctactaca gcaatgaaac cgttcccgtc tgccagcgctgctaccaggt ggtggagcgc 240 tacaaggaac gcgggtcagt gacaccggga tatgttgttcactctaccac catcagcgcc 300 acgccggctc gatcctcgcc ggtgccgcca cttcacacgactccagccct ccggccgcat 360 gcaccctcgc cgcagcccgc ttccgtcgtg tccacggcaacactcgtcca tcccgtagaa 420 gaagacgcag tgtccacaaa accctccgtc agcgaggccgacctgcacgc cctgcgaagc 480 atcatcgaga ccctccagca agcactcaac gatgaacaacacaacgccgc cctagccgcc 540 acgtccgctg ccgagcagct ccgaacagcc aaagaagagaacacagcact caaaagcacc 600 gcacatctcc tgcagcagcg cctagacacc gccacgcagcagcgcgccga gctggaggca 660 cgggtggcac ggctggccgc ggaccgcgac gaggcgcgccagcagctggc cgcgaacgcc 720 gaggagctgc agcagcgcct agacaccgcc acgcagcagcgcgccgagct ggaggcacgg 780 gtggcacggc tggccgcgga ccgcgacgag gcgcgccagcagctggccgc gaacgccgag 840 gagctgcagc agcgcctaga caccgccacg cagcagcgcgccgagctgga ggcacaggtg 900 gcacggctgg ccgcggacgg cgacgaggcg cgccagcagctggccgcgaa cgccgaggag 960 ctgcagcagc gcctagacac cgccacgcag cagcgcgccgagctggaggc acgggtggca 1020 cggctggccg cggaccgcga cgaggcgcgc cagcagctggccgcgaacgc cgaggagctg 1080 cagcagcgcc tagacaccgc cacgcagcag cgcgccgagctggaggcaca gttggcacgg 1140 ctggccgcgg accgcgacga ggcgcgccag cagctggccgcgaacgccga ggagctgcag 1200 cagcgcctag acaccgccac gcagcagcgc gccgagctggaggcacaggt ggcacggctg 1260 gccgcgaacg ccgaggagct gcagcagcgc ctagacaccgccacgcagca gcgcgccgag 1320 ctggaggcac gggtggcacg gctggccgcg gaccgcgacgaggcgcgcca gcagctggcc 1380 gcgaacgccg aggagctgca gcagcgccta gacaccgccacgcagcagcg cgccgagctg 1440 gaggcacggg tggcacggct ggccgcggac ggcgacgaggcccgccagca gctggccgcg 1500 aacgccgagg agctgcagca gcgcctagac accgccacgcagcagcgcgc cgagctggag 1560 gcacaggtgg cacggctggc cgcgaacgcc gaggagctgcagcagcgcct agacaccgcc 1620 acgcagcagc gcgccgagct ggaggcacgg gtggcacggctggccgcgga ccgcgacgag 1680 gcgcgccagc agctggccgc gaacgccgag gagctgcagcagcgcctaga caccgccacg 1740 cagcagcgcg ccgagctgga ggcacaggtg gcacggctggccgcgaacgc cgaggagctg 1800 cagcagcgcc tagacaccgc cacgcagcag cgcgccgagctggaggcacg ggtggcacgg 1860 ctggccgtgg accgcgacga ggcgcgccag cagctggccgcgaacgccga ggagctgcag 1920 cagcgcctag acaccgccac gcagcagcgc gccgagctggaggcacaggt ggcacggctg 1980 gccgcggacc gcgacgaggc gcgccagcag ctggccgcgaacgccgagga gctgcagcag 2040 cgcctagaca ccgccacgca gcagcgcgcc gagctggaggcacagttggc acggctggcc 2100 gcggaccgcg acgaggcgcg ccagcagctg gccgcgaacgccgaggagct gcagcagcgc 2160 ctagacaccg ccacgcagca gcgcgccgag ctggaggcacaggtggcacg gctggccgcg 2220 gaccgcgacg aggcgcgcca gcagctggcc gcgaacgccgaggagctgca gcagcgccta 2280 gacaccgcca cgcagcagcg cgccgagctg gaggcacagttggcacggct ggccgcggac 2340 cgcgacgagg cgcgccagca gctggccgcg aacgccgaggagctgcagca gcgcctagac 2400 accgccacgc agcagcgcgc cgagctggag gcacaggtggcacggctggc cgcggaccgc 2460 gacgaggcgc gccagcagct ggccgcgaac gccgaggagctgcagcagcg cctagacacc 2520 gccacgcagc agcgcgccga gctggaggca caggtggcacggctggccgc ggaccgcgac 2580 gaggcccgcc agcagctggc cgcgaacgcc gaggagctgcagcagcgcct agacaccgcc 2640 acgcagcagc gcgccgagct ggaggcacag gtggcacggctggccgcgaa cgccgaggag 2700 ctgcagcagc gcctagacac cgccacgcag cagcgcgccgagctggaggc acgggtggca 2760 cggctggccg cggaccgcga cgaggcgcgc cagcagctggccgcgaacgc cgaggagctg 2820 cagcagcgcc tagacaccgc cacgcagcag cgcgccgagctggaggcaca gttggcacgg 2880 ctggccgcgg accgcgacga ggcgcgccag cagctggccgcgaacgccga ggagctgcag 2940 cagcgcctag acaccgccac gcagcagcgc gccgagctggaggcacagtt ggcacggctg 3000 gccgcggacc gcgacgaggc gcgccagcag ctggccgcgaacgccgagga gctgcagcag 3060 cgcctagaca ccgccacgca gcagcgcgcc gagctggaggcacaggtggc acggctggcc 3120 gcggaccgcg acgaggcgcg ccagcagctg gccgcgaacgccgaggagct gcagcagcgc 3180 ctagacaccg ccacgcagca gcgcgccgag ctggaggcacgggtggcacg gctggccgcg 3240 gaccgcgacg aggcgcgcca gcagctggcc gcgaacgccgaggagctgca gcagcgccta 3300 gacaccgcca cgcagcagcg cgccgagctg gaggcacaggtggcacggct ggccgcggac 3360 ggcgacgagg cgcgccagca gctggccgcg aacgccgaggagctgcagca gcgcctagac 3420 accgccacgc agcagcgcgc cgagctggag gcacgggtggcacggctggc cgcggaccgc 3480 gacgaggcgc gccagcagct ggccgcgaac gccgaggagctgcagcagcg cctagacacc 3540 gccacgcagc agcgcgccga gctggaggca cagttggcacggctggccgc ggaccgcgac 3600 gaggcgcgcc agcagctggc cgcgaacgcc gaggagctgcagcagcgcct agacaccgcc 3660 acgcagcagc gcgccgagct ggaggcacag gtggcacggctggccgcgga cggcgacgag 3720 gcgcgccagc agctggccgc gaacgccgag gagctgcagcagcgcctaga caccgccacg 3780 cagcagcgcg ccgagctgga ggcacagttg gcacggctggccgcggaccg cgacgaggcg 3840 cgccagcagc tggccgcgaa cgccgaggag ctgcagcagcgcctagacac cgccacgcag 3900 cagcgcgccg agctggaggc acaggtggca cggctggccgcgaacgccga ggagctgcag 3960 cagcgcctag acaccgccac gcagcagcgc gccgagctggaggcacgggt ggcacggctg 4020 gccgcggacc gcgacgaggc gcgccagcag ctggccgcgaacgccgagga gctgcagcag 4080 cgcctagaca ccgccacgca gcagcgcgcc gagctggaggcacgggtggc acggctggcc 4140 gcggaccgcg acgaggcgcg ccagcagctg gccgcgaacgccgaggagct gcagcagcgc 4200 ctagacaccg ccacgcagca gcgcgccgag ctggaggcacaggtggcacg gctggccgcg 4260 aacgccgagg agctgcagca gcgcctagac accgccacgcagcagcgcgc cgagctggag 4320 gcacgggtgg cacggctggc cgcggaccgc gacgaggcgcgccagcagct ggccgcgaac 4380 gccgaggagc tgcagcagcg cctagacacc gccacgcagcagcgcgccga gctggaggca 4440 caggtggcac ggctggccgc ggaccgcgac gaggcgcgccagcagctggc cgcgaacgcc 4500 gaggagctgc agcagcgcct agacaccgcc acgcagcagcgcgccgagct ggaggcacgg 4560 gtggcacggc tggccgcgga cggcgacgag gcccgccagcagctggccgc gaacgccgag 4620 gagctgcagc agcgcctaga caccgccacg cagcagcgcgccgagctgga ggcacagttg 4680 gcacggctgg ccgcggaccg cgacgaggcg cgccagcagctggccgcgaa cgccgaggag 4740 ctgcagcagc gcctagacac cgccacgcag cagcgcgccgagctggaggc acgggtggca 4800 cggctggccg cggacggcga cgaggcgcgc cagcagctggccgcgaacgc cgaggagctg 4860 cagcagcgcc tagacaccgc cacgcagcag cgcgccgagctggaggcacg ggtggcacgg 4920 ctggccgcgg accgcgacga ggcgcgccag cagctggccgcgaacgccga ggagctgcag 4980 cagcgcctag acaccgccac gcagcagcgc gccgagctggaggcacagtt ggcacggctg 5040 gccgcggacc gcgacgaggc gcgccagcag ctggccgcgaacgccgagga gctgcagcag 5100 cgcctagaca ccgccacgca gcagcgcgcc gagctggaggcacagttggc acggctggcc 5160 gcggacggcg acgaggcgcg ccagcagctg gccgcgaacgccgaggagct gcagcagcgc 5220 ctagacaccg ccacgcagca gcgcgccgag ctggaggtggagatggcagt tctcctgcgt 5280 gagagggagg aagctcgcgg agagacagca gtggctggagagcaggtgca gctgtaccgg 5340 gaaacggtcg aggaggagga gtgtttaaag gaggaacgatggtgcctcga gtcacgggtg 5400 gcgcagctga gggaggcgtc cgcggccgcg aagcagcagcggcaagaagt ggcggcgaag 5460 gcgaatgagg tgcaggagcg ccttgattcg atggcgcgtcggtgcattgc gcatgaggga 5520 gatgcgccgc agcgggctga cgggagagac gacgccttgcggcaacttgc taatctgcgt 5580 gaagaggtga agctcagtga gaagcagaag gcgatggagcgtgttattcc gggtgttaga 5640 gagcggcaga tgcgcctcga agcagctgag gagcagcgggcggacttgga agcacgcctc 5700 gtcgacgagg ccggtgacct gcgctctcgt cctgctgcgagcacaaacga agtgaatctg 5760 taccgcgacc tcgctctgca ggaacacgaa gcagcccagaatcggtgcac aacactggaa 5820 gcgcaggtgg caagcctcac gagcgaccgc gacaacgggcgccagcaaga gtcggctgac 5880 ctcagcgagg cgcagcgtca cctcgacaac gtgcaggagcgcgacatggc ccatcatcgg 5940 tgtgccgcgt tggaggagca gaatgctgct atggcctcggagttgcaagc ggtcaaggct 6000 aaactgcggc aggcctcagt gaaggcctcc tcgttgatgacgcgactctc tgcatcatcc 6060 tcgggagctg gtggtgtctc tgcccgtgtg agagtgggaggcagctctgc cgtgccacaa 6120 gcggcgccac accgggatgc ggagctcatc gccgaggtgggcgaacgcct tcgcgaaagg 6180 ggggaggcca tgcgactgtt ggccgaaggc gtcgaactgcgcgagcgcgc gcggccgctg 6240 gaacgggtgc tcgccgagaa gctgatcggc gaccgtcgcacgagcgacgc ggaagaggtc 6300 gccacggagc ccacgcaggt gcggcggaac gctgcgcactcgcggcacct ggactcgcgt 6360 gaggcgcagc tggacgagcg cgcggcacgg ctgagggaaaaggagcagca gctgctgcgc 6420 gtcgctcgtg aactgcagac taagtcgcgc gcccttcaagtattatacgc acgggcgctg 6480 aataggccac aggtgacgtc gttgctgctg acggccgacggcgacgatac ctcctacccc 6540 gacacaccac agcaacagca gcaaggcaca cgcacgccgctcagagaacc tgtatattcg 6600 ctggacagcg aggtggcgca ctacggccga actgcaggggccgcagtgag cagtggtctc 6660 gcatcacctc tgccgaggga gccgccacgg gctcggatggtgcaccgcgc tgtggaggca 6720 acgggcacag aggaagacac gcaggtgcgc ctcacggctgcaacggaggc ttaccgcgac 6780 gtactgtacg agcacattct tgagtcgaat gggctccaaggggtggatgt gttggctcag 6840 tacctgcccc accacacctc cggcggtggg ctgaagacaccgcgactgcc ggggagcggc 6900 attatatcga agacccgggc aatgctacga gcgctggaggagcgcctggg tgcttcccgt 6960 ggcgtcgggc gtggagtcga cccggcggtg caggagcgaagcctggaagc gttccggcgg 7020 ctcgaggccg ccttgtctgc tctctgtgga ggcagccatgcgtag 7065 <210> SEQ ID NO 116 <211> LENGTH: 2589 <212> TYPE: DNA <213>ORGANISM: Leishmania major and chagasi <220> FEATURE: <221> NAME/KEY:misc_feature <222> LOCATION: (1)...(2589) <223> OTHER INFORMATION: n =A,T,C or G <400> SEQUENCE: 116 gtmccttcca agcttgcaat ccgtctcatgcgcagctctc gcggcggctc tctcgcggtg 60 gtcgccttgg cggtttgcct ggcagtgctcgccgcgatcg gcacgtgtgt tttcgactcg 120 caagagatcg gcggcagctc cttcaccttcgtaggctggt cgtctgccag caaggaggaa 180 tcctaccagg gctgcaccct gaccgggaaggcattcagaa ttcaaggcgc tgcaagctct 240 ctgtcggacg acgccacgct gcccggtgggatcctgcggt tctccagcct gcttgtatcg 300 aacggctaca tcgtcgtgga taagtactttccccggaaca ccaacatcac catcaaagat 360 gccagcggca ccgtcgctgc ggggatgcctttcatcgacg ccaacacggc gatatactct 420 gatcagctca gcatcgtggt caccgactccactctcagtt gggctgccgc acggtcgggt 480 cagagcatgg tgcgtgcacc cttcactattcagctctcca gctctctctt cgtgctcggc 540 agcaccgtga cgcaggcctc gtcagtggtagaggtggtgg gcccgagcag catctcccag 600 aagtctgcat tggctgtgga ctacgccaagtgcaccggct gtgctcaggg tctggtgcac 660 ttcaccgact ttgtcagagt gtgggaccgcagcttgcttc gcgtctccca ctccagcgtc 720 aagggcgcca ccggcaagcc gctgatcggcatagcacaga gcgcaggggc gagcctggcg 780 gtggaaaaca gtctgttcgt tgtcgagaacgtgtcatcgc ccaccagtaa cctcatcgac 840 gcggccgtca gaatgggcac cgatgcgcagatcaccctac gcgctgtgac ggtgaagagc 900 attggtgcaa cgatggccgg cagcgtcactgcgcagctct tgacggcaga cgacatcgcc 960 caacaaattc cctccatttc cgtggtgccagacacccgct gcgctgcggc ctgcgtcccc 1020 acggccacgg tcgactccag ctgcaagtgtgcctgcaatg ccgacatgcc gaacatgaac 1080 ttctgcacgg ccatgaagga cccctacacaaactatgcct acctcggctg ctcggcgggc 1140 tgcacaacgt gcttcaacga gaccgcctgcctcgagtgca ggccgagcta cgagatgctg 1200 cccgacatga catgctcgct gaccggacttcagtgcacag acccgaactg caagacctgc 1260 acaacttacg gtcagtgcac agactgcaacgacggctacg gtctcacctc ctccagcgtt 1320 tgcgtgcgct gcagtgtagc gggctgcaagagctgccccg tcgacgctaa cgtctgcaaa 1380 gtgtgtctcg gcggcagcga gccgatcaacaatatgtgcc cctgcaccga ccccaactgc 1440 gccagctgcc ccagcgacgc tggcacgtgcactcagtgcg cgaacggcta cggtctcgtg 1500 gacggcgcct gtgtgagatg ccaggagcccaactgcttca gctgcgacag cgacgcgaat 1560 aagtgcacac aatgtgcgcc gaactactacctcaccccgc tcttgacctg ctccccggtg 1620 gcctgcaaca tcgagcactg catgcagtgcgacccacaga cgccgtcgcg ctgccaggag 1680 tgcgtgtccc cctacgtggt tgacagctacgacggcctct gcaggctctc cgatgcctgc 1740 tccgtgccca actgcaagaa gtgcgagaccggtacctcca ggctctgcgc cgagtgcgac 1800 accggctaca gtctctccgc cgacgcgacgagctgcagca gtccaaccac gcagccgtgc 1860 gaggtggagc actgcaacac atgtgtgaacggcgatagca cccgctgtgc ctactgcaac 1920 accggctact acgtctccga tggcaagtgcaaggccatgc agggctgcta cgtgtcgaac 1980 tgcgcgcagt gcatgctgct tgacagcaccaagtgctcca cgtgcgtgaa agggtacctg 2040 ctcacgtcgt cctacagttg cgtctcgcagaaagtcatca acagtgcggc cgcgccctac 2100 tctctgtggg tggccgccgc cgtgctcctcacctcttttg ccatgcacct agcataggct 2160 agcccgggac gcgtggatcc tcgcaatccctaggaggatt aggcaagggc ttgagctcac 2220 gctcttgtga gggacagaaa tacaatcaggggcagtatat gaatactcca tggagaaacc 2280 cagatctacg tatgatcagc ctcgactgtgccttctagtt gccagccatc tgttgtttgc 2340 ccctcccccg tgccttcctt gaccctggaaggtgccactc ccactgtcct ttcctaataa 2400 aatgaggaaa ttgcatcgca ttgtctgagtaggtgtcatt ctattctggg gggtggggtg 2460 gggcangaca gcaaggggga ggattgggaagacaatagca ggcatgctgg ggatgcggtg 2520 ggctctatgg cttctgaggc ggaaagaaccagctggggct cgacagctcg agctaggacc 2580 gctatcang 2589 <210> SEQ ID NO 117<211> LENGTH: 909 <212> TYPE: DNA <213> ORGANISM: Leishmania major andchagasi <400> SEQUENCE: 117 atgcatcacc atcaccatca cgactcgcaa gagatcggcggcagctcctt caccttcgta 60 ggctggtcgt ctgccagcaa ggaggaatcc taccagggctgcaccctgac cgggaaggca 120 ttcagaattc aaggcgctgc aagctctctg tcggacgacgccacgctgcc cggtgggatc 180 ctgcggttct ccagcctgct tgtatcgaac ggctacatcgtcgtggataa gtactttccc 240 cggaacacca acatcaccat caaagatgcc agcggcaccgtcgctgcggg gatgcctttc 300 atcgacgcca acacggcgat atactctgat cagctcagcatcgtggtcac cgactccact 360 ctcagttggg ctgccgcacg gtcgggtcag agcatggtgcgtgcaccctt cactattcag 420 ctctccagct ctctcttcgt gctcggcagc accgtgacgcaggcctcgtc agtggtagag 480 gtggtgggcc cgagcagcat ctcccagaag tctgcattggctgtggacta cgccaagtgc 540 accggctgtg ctcagggtct ggtgcacttc accgactttgtcagagtgtg ggaccgcagc 600 ttgcttcgcg tctcccactc cagcgtcaag ggcgccaccggcaagccgct gatcggcata 660 gcacagagcg caggggcgag cctggcggtg gaaaacagtctgttcgttgt cgagaacgtg 720 tcatcgccca ccagtaacct catcgacgcg gccgtcagaatgggcaccga tgcgcagatc 780 accctacgcg ctgtgacggt gaagagcatt ggtgcaacgatggccggcag cgtcactgcg 840 cagctcttga cggcagacga catcgcccaa caaattccctccatttccgt ggtgccagac 900 acccgctaa 909 <210> SEQ ID NO 118 <211>LENGTH: 538 <212> TYPE: PRT <213> ORGANISM: Leishmania major and chagasi<400> SEQUENCE: 118 Arg Asn Thr Tyr Leu Leu Pro Leu Leu Gly Arg Arg ThrThr Thr Thr 5 10 15 Phe Lys Thr Thr Pro Arg Leu Leu Val Pro His Leu LeuLeu Ser Thr 20 25 30 Phe Asn Pro Cys Leu Ala Asn Met Glu Ala Tyr Lys LysLeu Glu Thr 35 40 45 Ile Phe Thr Lys Val Tyr Arg Leu Asp His Phe Leu GlyLeu Gly Asn 50 55 60 Trp Asp Met Asn Thr Asn Met Pro Pro Lys Gly Glu GluSer Arg Gly 65 70 75 80 Glu Ala Met Ala Met Leu Ser Glu Leu Arg Phe GlyPhe Ile Thr Ala 85 90 95 Pro Glu Val Lys Ser Leu Ile Glu Ser Ala Thr LysGly Ser Glu Glu 100 105 110 Leu Asn Ala Val Gln Arg Ala Asn Leu Arg GluMet Arg Arg Ala Trp 115 120 125 Lys Ser Ala Thr Ala Leu Pro Ala Glu PheVal Gly Arg Lys Met Arg 130 135 140 Leu Thr Thr His Ala His Ser Val TrpArg Asp Ser Arg Lys Ala Asn 145 150 155 160 Asp Phe Ala Lys Phe Leu ProVal Leu Arg Asp Leu Val Ala Leu Ala 165 170 175 Arg Glu Glu Gly Ser TyrLeu Ala Ala Gly Thr Ser Leu Ser Pro Tyr 180 185 190 Glu Ala Leu Met AsnGlu Tyr Glu Pro Gly Ile Thr Thr Gln Lys Leu 195 200 205 Asp Glu Val TyrAla Asn Val Lys Ser Trp Leu Pro Gln Leu Leu Lys 210 215 220 Asp Ile ValGln Lys Gln Ser Gly Glu Ser Val Ile Ala Phe Ser His 225 230 235 240 LysPhe Pro Gln Asp Lys Gln Glu Ala Leu Cys Lys Glu Phe Met Lys 245 250 255Ile Trp His Phe Asp Thr Asp Ala Gly Arg Leu Asp Val Ser Pro His 260 265270 Pro Phe Thr Gly Met Thr Lys Glu Asp Cys Arg Leu Thr Thr Asn Tyr 275280 285 Ile Glu Asp Thr Phe Val Gln Ser Leu Tyr Gly Val Ile His Glu Ser290 295 300 Gly His Gly Lys Tyr Glu Gln Asn Cys Gly Pro Arg Glu His IleThr 305 310 315 320 Gln Pro Val Cys Asn Ala Arg Ser Leu Gly Leu His GluSer Gln Ser 325 330 335 Leu Phe Ala Glu Phe Gln Ile Gly His Ala Thr ProPhe Ile Asp Tyr 340 345 350 Leu Thr Thr Arg Leu Pro Glu Phe Phe Glu AlaGln Pro Ala Phe Ser 355 360 365 Gln Asp Asn Met Arg Lys Ser Leu Gln GlnVal Lys Pro Gly Tyr Ile 370 375 380 Arg Val Asp Ala Asp Glu Val Cys TyrPro Leu His Val Ile Leu Arg 385 390 395 400 Tyr Glu Ile Glu Arg Asp LeuMet Glu Gly Lys Met Glu Val Glu Asp 405 410 415 Val Pro Arg Ala Trp AsnAla Lys Met Gln Glu Tyr Leu Gly Leu Ser 420 425 430 Thr Glu Gly Arg AspAsp Val Gly Cys Leu Gln Asp Val His Trp Ser 435 440 445 Met Gly Ala LeuGly Tyr Phe Pro Thr Tyr Ser Leu Gly Ala Met Tyr 450 455 460 Ala Ala GlnIle Met Ala Ser Ile Arg Lys Glu Leu Gly Asp Asp Lys 465 470 475 480 ValAsp Glu Cys Leu Arg Thr Gly Glu Leu Gly Pro Leu Leu Glu Lys 485 490 495Gln Gln Glu Lys Ile Trp Asp His Gly Cys Leu Tyr Glu Thr Asp Asp 500 505510 Leu Met Thr Arg Ala Thr Gly Glu Thr Leu Asn Pro Glu Tyr Leu Arg 515520 525 Arg His Leu Glu Ala Arg Tyr Leu Asn Ala 530 535 <210> SEQ ID NO119 <211> LENGTH: 193 <212> TYPE: PRT <213> ORGANISM: Leishmania majorand chagasi <400> SEQUENCE: 119 Met His His His His His His Asp Pro ProGly Cys Arg Asn Ser Ala 5 10 15 Arg Glu Leu Gln Gln Arg Leu Asp Thr AlaThr Gln Gln Arg Ala Glu 20 25 30 Leu Glu Ala Arg Val Ala Arg Leu Ala AlaAsp Arg Asp Glu Ala Arg 35 40 45 Gln Gln Leu Ala Ala Asn Ala Glu Glu LeuGln Gln Arg Leu Asp Thr 50 55 60 Ala Thr Gln Gln Arg Ala Glu Leu Glu AlaArg Val Ala Arg Leu Ala 65 70 75 80 Ala Asp Gly Asp Glu Ala Arg Gln GlnLeu Ala Ala Asn Ala Glu Glu 85 90 95 Leu Gln Gln Arg Leu Asp Thr Ala ThrGln Gln Arg Ala Glu Leu Glu 100 105 110 Ala Gln Val Ala Arg Leu Ala AlaAsn Ala Glu Glu Leu Gln Gln Arg 115 120 125 Leu Asp Thr Ala Thr Gln GlnArg Ala Glu Leu Glu Ala Arg Val Ala 130 135 140 Arg Leu Ala Ala Asp ArgAsp Glu Ala Arg Gln Gln Leu Ala Ala Asn 145 150 155 160 Ala Glu Glu LeuGln Gln Arg Leu Asp Thr Ala Thr Gln Gln Arg Ala 165 170 175 Glu Leu GluAla Gln Val Ala Arg Leu Ala Ala Asn Ala Ala Arg Ala 180 185 190 Glu<210> SEQ ID NO 120 <211> LENGTH: 2310 <212> TYPE: PRT <213> ORGANISM:Leishmania major and chagasi <400> SEQUENCE: 120 Met Ser Thr Pro Val SerGly Val Val Pro Gln Asp Arg Trp Gln Pro 5 10 15 Gln Gln Arg Val Lys ValCys Gln Tyr Gln Asp Cys Gly Ala Pro Phe 20 25 30 Gly Phe Phe Ser Thr LysVal Asn Cys His Arg Cys Gly Ile Val Leu 35 40 45 Cys Ser Lys Cys Ala AlaThr Lys Thr Val Ile Pro Arg Tyr Tyr Ser 50 55 60 Asn Glu Thr Val Pro ValCys Gln Arg Cys Tyr Gln Val Val Glu Arg 65 70 75 80 Tyr Lys Glu Arg GlySer Val Thr Pro Gly Tyr Val Val His Ser Thr 85 90 95 Thr Ile Ser Ala ThrPro Ala Arg Ser Ser Pro Val Pro Pro Leu His 100 105 110 Thr Thr Pro AlaLeu Arg Pro His Ala Pro Ser Pro Gln Pro Ala Ser 115 120 125 Val Val SerThr Ala Thr Leu Val His Pro Val Glu Glu Asp Ala Val 130 135 140 Ser ThrLys Pro Ser Val Ser Glu Ala Asp Leu His Ala Leu Arg Ser 145 150 155 160Ile Ile Glu Thr Leu Gln Gln Ala Leu Asn Asp Glu Gln His Asn Ala 165 170175 Ala Leu Ala Ala Thr Ser Ala Ala Glu Gln Leu Arg Thr Ala Lys Glu 180185 190 Glu Asn Thr Ala Leu Lys Ser Thr Ala His Leu Leu Gln Gln Arg Leu195 200 205 Asp Thr Ala Thr Gln Gln Arg Ala Glu Leu Glu Ala Gln Val AlaArg 210 215 220 Leu Ala Ala Asp Arg Asp Glu Ala Arg Gln Gln Leu Ala AlaAsn Ala 225 230 235 240 Glu Glu Leu Gln Gln Arg Leu Asp Thr Ala Thr GlnGln Arg Ala Glu 245 250 255 Leu Glu Ala Arg Val Ala Arg Leu Ala Ala AspArg Asp Glu Ala Arg 260 265 270 Gln Gln Leu Ala Ala Asn Ala Glu Glu LeuGln Gln Arg Leu Asp Thr 275 280 285 Ala Thr Gln Gln Arg Ala Glu Leu GluAla Gln Val Ala Arg Leu Ala 290 295 300 Ala Asn Ala Glu Glu Leu Gln GlnArg Leu Asp Thr Ala Thr Gln Gln 305 310 315 320 Arg Ala Glu Leu Glu AlaGln Leu Ala Arg Leu Ala Ala Asp Arg Asp 325 330 335 Glu Ala Arg Gln GlnLeu Ala Ala Asn Ala Glu Glu Leu Gln Gln Arg 340 345 350 Leu Asp Thr AlaThr Gln Gln Arg Ala Glu Leu Glu Ala Gln Val Ala 355 360 365 Arg Leu AlaAla Asn Ala Glu Glu Leu Gln Gln Arg Leu Asp Thr Ala 370 375 380 Thr GlnGln Arg Ala Glu Leu Glu Ala Arg Val Ala Arg Leu Ala Ala 385 390 395 400Asp Arg Asp Glu Ala Arg Gln Gln Leu Ala Ala Asn Ala Glu Glu Leu 405 410415 Gln Gln Arg Leu Asp Thr Ala Thr Gln Gln Arg Ala Glu Leu Glu Ala 420425 430 Gln Val Ala Arg Leu Ala Ala Asn Arg Asp Glu Ala Arg Gln Gln Leu435 440 445 Ala Ala Asn Ala Glu Glu Leu Gln Gln Arg Leu Asp Thr Ala ThrGln 450 455 460 Gln Arg Ala Glu Leu Glu Ala Gln Val Ala Arg Leu Ala AlaAsp Arg 465 470 475 480 Asp Glu Ala Arg Gln Gln Leu Ala Ala Asn Ala GluGlu Leu Gln Gln 485 490 495 Arg Leu Asp Thr Ala Thr Gln Gln Arg Ala GluLeu Glu Ala Arg Val 500 505 510 Ala Arg Leu Ala Ala Asp Arg Asp Glu AlaArg Gln Gln Leu Ala Ala 515 520 525 Asn Ala Glu Glu Leu Gln Gln Arg LeuAsp Thr Ala Thr Gln Gln Arg 530 535 540 Ala Glu Leu Glu Ala Arg Val AlaArg Leu Ala Ala Asn Ala Glu Glu 545 550 555 560 Leu Gln Gln Arg Leu AspThr Ala Thr Gln Gln Arg Ala Glu Leu Glu 565 570 575 Ala Gln Val Ala ArgLeu Ala Ala Asn Ala Glu Glu Leu Gln Gln Arg 580 585 590 Leu Asp Thr AlaThr Gln Gln Arg Ala Glu Leu Glu Ala Arg Val Ala 595 600 605 Arg Leu AlaAla Asp Arg Asp Glu Ala Arg Gln Gln Leu Ala Ala Asn 610 615 620 Ala GluGlu Leu Gln Gln Arg Leu Asp Thr Ala Thr Gln Gln Arg Ala 625 630 635 640Glu Leu Glu Ala Gln Leu Ala Arg Leu Ala Ala Asp Gly Asp Glu Ala 645 650655 Arg Gln Gln Leu Ala Ala Asn Ala Glu Glu Leu Gln Gln Arg Leu Asp 660665 670 Thr Ala Thr Gln Gln Arg Ala Glu Leu Glu Ala Arg Val Ala Arg Leu675 680 685 Ala Ala Asp Arg Asp Glu Ala Arg Gln Gln Leu Ala Ala Asn AlaGlu 690 695 700 Glu Leu Gln Gln Arg Leu Asp Thr Ala Thr Gln Gln Arg AlaGlu Leu 705 710 715 720 Glu Ala Gln Leu Ala Arg Leu Ala Ala Asp Arg AspGlu Ala Arg Gln 725 730 735 Gln Leu Ala Ala Asn Ala Glu Glu Leu Gln GlnArg Leu Asp Thr Ala 740 745 750 Thr Gln Gln Arg Ala Glu Leu Glu Ala ArgVal Ala Arg Leu Ala Ala 755 760 765 Asp Gly Asp Glu Ala Arg Gln Gln LeuAla Ala Asn Ala Glu Glu Leu 770 775 780 Gln Gln Arg Leu Asp Thr Ala ThrGln Gln Arg Ala Glu Leu Glu Ala 785 790 795 800 Arg Val Ala Arg Leu AlaAla Asp Arg Asp Glu Ala Arg Gln Gln Leu 805 810 815 Ala Ala Asn Ala GluGlu Leu Gln Gln Arg Leu Asp Thr Ala Thr Gln 820 825 830 Gln Arg Ala GluLeu Glu Ala Gln Leu Ala Arg Leu Ala Ala Asp Gly 835 840 845 Asp Glu AlaArg Gln Gln Leu Ala Ala Asn Ala Glu Glu Leu Gln Gln 850 855 860 Arg LeuAsp Thr Ala Thr Gln Gln Arg Ala Glu Leu Glu Ala Arg Val 865 870 875 880Ala Arg Leu Ala Ala Asp Arg Asp Glu Ala Arg Gln Gln Leu Ala Ala 885 890895 Asn Ala Glu Glu Leu Gln Gln Arg Leu Asp Thr Ala Thr Gln Gln Arg 900905 910 Ala Glu Leu Glu Ala Gln Leu Ala Arg Leu Ala Ala Asp Arg Asp Glu915 920 925 Ala Arg Gln Gln Leu Ala Ala Asn Ala Glu Glu Leu Gln Gln ArgLeu 930 935 940 Asp Thr Ala Thr Gln Gln Arg Ala Glu Leu Glu Ala Gln ValAla Arg 945 950 955 960 Leu Ala Ala Asp Gly Asp Glu Ala Arg Gln Gln LeuAla Ala Asn Ala 965 970 975 Glu Glu Leu Gln Gln Arg Leu Asp Thr Ala ThrGln Gln Arg Ala Glu 980 985 990 Leu Glu Ala Gln Leu Ala Arg Leu Ala AlaAsp Arg Asp Glu Ala Arg 995 1000 1005 Gln Gln Leu Ala Ala Asn Ala GluGlu Leu Gln Gln Arg Leu Asp Thr 1010 1015 1020 Ala Thr Gln Gln Arg AlaGlu Leu Glu Ala Gln Val Ala Arg Leu Ala 1025 1030 1035 1040 Ala Asn AlaGlu Glu Leu Gln Gln Arg Leu Asp Thr Ala Thr Gln Gln 1045 1050 1055 ArgAla Glu Leu Glu Ala Arg Val Ala Arg Leu Ala Ala Asp Arg Asp 1060 10651070 Glu Ala Arg Gln Gln Leu Ala Ala Asn Ala Glu Glu Leu Gln Gln Arg1075 1080 1085 Leu Asp Thr Ala Thr Gln Gln Arg Ala Glu Leu Glu Ala ArgVal Ala 1090 1095 1100 Arg Leu Ala Ala Asn Ala Glu Glu Leu Gln Gln ArgLeu Asp Thr Ala 1105 1110 1115 1120 Thr Gln Gln Arg Ala Glu Leu Glu AlaGln Val Ala Arg Leu Ala Ala 1125 1130 1135 Asn Ala Glu Glu Leu Gln GlnArg Leu Asp Thr Ala Thr Gln Gln Arg 1140 1145 1150 Ala Glu Leu Glu AlaArg Val Ala Arg Leu Ala Ala Asp Arg Asp Glu 1155 1160 1165 Ala Arg GlnGln Leu Ala Ala Asn Ala Glu Glu Leu Gln Gln Arg Leu 1170 1175 1180 AspThr Ala Thr Gln Gln Arg Ala Glu Leu Glu Ala Gln Val Ala Arg 1185 11901195 1200 Leu Ala Ala Asn Ala Glu Glu Leu Gln Gln Arg Leu Asp Thr AlaThr 1205 1210 1215 Gln Gln Arg Ala Glu Leu Glu Ala Gln Leu Ala Arg LeuAla Ala Asp 1220 1225 1230 Arg Asp Glu Ala Arg Gln Gln Leu Ala Ala AsnAla Glu Glu Leu Gln 1235 1240 1245 Gln Arg Leu Asp Thr Ala Thr Gln GlnArg Ala Glu Leu Glu Ala Gln 1250 1255 1260 Val Ala Arg Leu Ala Ala AsnAla Glu Glu Leu Gln Gln Arg Leu Asp 1265 1270 1275 1280 Thr Ala Thr GlnGln Arg Ala Glu Leu Glu Ala Arg Val Ala Arg Leu 1285 1290 1295 Ala AlaAsp Arg Asp Glu Ala Arg Gln Gln Leu Ala Ala Asn Ala Glu 1300 1305 1310Glu Leu Gln Gln Arg Leu Asp Thr Ala Thr Gln Gln Arg Ala Glu Leu 13151320 1325 Glu Ala Arg Val Ala Arg Leu Ala Ala Asp Gly Asp Glu Ala ArgGln 1330 1335 1340 Gln Leu Ala Ala Asn Ala Glu Glu Leu Gln Gln Arg LeuAsp Thr Ala 1345 1350 1355 1360 Thr Gln Gln Arg Ala Glu Leu Glu Ala GlnVal Ala Arg Leu Ala Ala 1365 1370 1375 Asn Ala Glu Glu Leu Gln Gln ArgLeu Asp Thr Ala Thr Gln Gln Arg 1380 1385 1390 Ala Glu Leu Glu Ala ArgVal Ala Arg Leu Ala Ala Asp Arg Asp Glu 1395 1400 1405 Ala Arg Gln GlnLeu Ala Ala Asn Ala Glu Glu Leu Gln Gln Arg Leu 1410 1415 1420 Asp ThrAla Thr Gln Gln Arg Ala Glu Leu Glu Ala Gln Val Ala Arg 1425 1430 14351440 Leu Ala Ala Asp Arg Asp Glu Ala Arg Gln Gln Leu Ala Ala Asn Ala1445 1450 1455 Glu Glu Leu Gln Gln Arg Leu Asp Thr Ala Thr Gln Gln ArgAla Glu 1460 1465 1470 Leu Glu Ala Arg Val Ala Arg Leu Ala Ala Asp GlyAsp Glu Ala Arg 1475 1480 1485 Gln Gln Leu Ala Ala Asn Ala Glu Glu LeuGln Gln Arg Leu Asp Thr 1490 1495 1500 Ala Thr Gln Gln Arg Ala Glu LeuGlu Ala Gln Leu Ala Arg Leu Ala 1505 1510 1515 1520 Ala Asp Arg Asp GluAla Arg Gln Gln Leu Ala Ala Asn Ala Glu Glu 1525 1530 1535 Leu Gln GlnArg Leu Asp Thr Ala Thr Gln Gln Arg Ala Glu Leu Glu 1540 1545 1550 AlaArg Val Ala Arg Leu Ala Ala Asp Gly Asp Glu Ala Arg Gln Gln 1555 15601565 Leu Ala Ala Asn Ala Glu Glu Leu Gln Gln Arg Leu Asp Thr Ala Thr1570 1575 1580 Gln Gln Arg Ala Glu Leu Glu Ala Arg Val Ala Arg Leu AlaAla Asp 1585 1590 1595 1600 Arg Asp Glu Ala Arg Gln Gln Leu Ala Ala AsnAla Glu Glu Leu Gln 1605 1610 1615 Gln Arg Leu Asp Thr Ala Thr Gln GlnArg Ala Glu Leu Glu Ala Gln 1620 1625 1630 Leu Ala Arg Leu Ala Ala AspArg Asp Glu Ala Arg Gln Gln Leu Ala 1635 1640 1645 Ala Asn Ala Glu GluLeu Gln Gln Arg Leu Asp Thr Ala Thr Gln Gln 1650 1655 1660 Arg Ala GluLeu Glu Ala Gln Leu Ala Arg Leu Ala Ala Asp Gly Asp 1665 1670 1675 1680Glu Ala Arg Gln Gln Leu Ala Ala Asn Ala Glu Glu Leu Gln Gln Arg 16851690 1695 Leu Asp Thr Ala Thr Gln Gln Arg Ala Glu Leu Glu Val Glu MetAla 1700 1705 1710 Val Leu Leu Arg Glu Arg Glu Glu Ala Arg Gly Glu ThrAla Val Ala 1715 1720 1725 Gly Glu Gln Val Gln Leu Tyr Arg Glu Thr ValGlu Glu Glu Glu Cys 1730 1735 1740 Leu Lys Glu Glu Arg Trp Cys Leu GluSer Arg Val Ala Gln Leu Arg 1745 1750 1755 1760 Glu Ala Ser Ala Ala AlaLys Gln Gln Arg Gln Glu Val Ala Ala Lys 1765 1770 1775 Ala Asn Glu ValGln Glu Arg Leu Asp Ser Met Ala Arg Arg Cys Ile 1780 1785 1790 Ala HisGlu Gly Asp Ala Pro Gln Arg Ala Asp Gly Arg Asp Asp Ala 1795 1800 1805Leu Arg Gln Leu Ala Asn Leu Arg Glu Glu Val Lys Leu Ser Glu Lys 18101815 1820 Gln Lys Ala Met Glu Arg Val Ile Pro Gly Val Arg Glu Arg GlnMet 1825 1830 1835 1840 Arg Leu Glu Ala Ala Glu Glu Gln Arg Ala Asp LeuGlu Ala Arg Leu 1845 1850 1855 Val Asp Glu Ala Gly Asp Leu Arg Ser ArgPro Ala Ala Ser Thr Asn 1860 1865 1870 Glu Val Asn Leu Tyr Arg Asp LeuAla Leu Gln Glu His Glu Ala Ala 1875 1880 1885 Gln Asn Arg Cys Thr ThrLeu Glu Ala Gln Val Ala Ser Leu Thr Ser 1890 1895 1900 Asp Arg Asp AsnGly Arg Gln Gln Glu Ser Ala Asp Leu Ser Glu Ala 1905 1910 1915 1920 GlnArg His Leu Asp Asn Val Gln Glu Arg Asp Met Ala His His Arg 1925 19301935 Cys Ala Ala Leu Glu Glu Gln Asn Ala Ala Met Ala Ser Glu Leu Gln1940 1945 1950 Ala Val Lys Ala Lys Leu Arg Gln Ala Ser Val Lys Ala SerSer Leu 1955 1960 1965 Met Thr Arg Leu Ser Ala Ser Ser Ser Gly Ala GlyGly Val Ser Ala 1970 1975 1980 Arg Val Arg Val Gly Gly Ser Ser Ala ValPro Gln Ala Ala Pro His 1985 1990 1995 2000 Arg Asp Ala Glu Leu Ile AlaGlu Val Gly Glu Arg Leu Arg Glu Arg 2005 2010 2015 Gly Glu Ala Met ArgLeu Leu Ala Glu Gly Val Glu Leu Arg Glu Arg 2020 2025 2030 Ala Arg ProLeu Glu Arg Val Leu Ala Glu Lys Leu Ile Gly Asp Arg 2035 2040 2045 ArgThr Ser Asp Ala Glu Glu Val Ala Thr Glu Pro Thr Gln Val Arg 2050 20552060 Arg Asn Ala Ala His Ser Arg His Leu Asp Ser Arg Glu Ala Gln Leu2065 2070 2075 2080 Asp Glu Arg Ala Ala Arg Leu Arg Glu Lys Glu Gln GlnLeu Leu Arg 2085 2090 2095 Val Ala Arg Glu Leu Gln Thr Lys Ser Arg AlaLeu Gln Val Leu Tyr 2100 2105 2110 Ala Arg Ala Leu Asn Arg Pro Gln ValThr Ser Leu Leu Leu Thr Ala 2115 2120 2125 Asp Gly Asp Asp Thr Ser TyrPro Asp Thr Pro Gln Gln Gln Gln Gln 2130 2135 2140 Gly Thr Arg Thr ProLeu Arg Glu Pro Val Tyr Ser Leu Asp Ser Glu 2145 2150 2155 2160 Val AlaHis Tyr Gly Arg Thr Ala Gly Ala Ala Val Ser Ser Gly Leu 2165 2170 2175Ala Ser Pro Leu Pro Arg Glu Pro Pro Arg Ala Arg Met Val His Arg 21802185 2190 Ala Val Glu Ala Thr Gly Thr Glu Glu Asp Thr Gln Val Arg LeuThr 2195 2200 2205 Ala Ala Thr Glu Ala Tyr Arg Asp Val Leu Tyr Glu HisIle Leu Glu 2210 2215 2220 Ser Asn Gly Leu Gln Gly Val Asp Val Leu AlaGln Tyr Leu Pro His 2225 2230 2235 2240 His Thr Ser Gly Gly Gly Leu LysThr Pro Arg Leu Pro Gly Ser Gly 2245 2250 2255 Ile Ile Ser Lys Thr ArgAla Met Leu Arg Ala Leu Glu Glu Arg Leu 2260 2265 2270 Gly Ala Ser ArgGly Val Gly Arg Gly Val Asp Pro Ala Val Gln Glu 2275 2280 2285 Arg SerLeu Glu Ala Phe Arg Arg Leu Glu Ala Ala Leu Ser Ala Leu 2290 2295 2300Cys Gly Gly Ser His Ala 2305 2310 <210> SEQ ID NO 121 <211> LENGTH: 709<212> TYPE: PRT <213> ORGANISM: Leishmania major and chagasi <400>SEQUENCE: 121 Met Arg Ser Ser Arg Gly Gly Ser Leu Ala Val Val Ala LeuAla Val 5 10 15 Cys Leu Ala Val Leu Ala Ala Ile Gly Thr Cys Val Phe AspSer Gln 20 25 30 Glu Ile Gly Gly Ser Ser Phe Thr Phe Val Gly Trp Ser SerAla Ser 35 40 45 Lys Glu Glu Ser Tyr Gln Gly Cys Thr Leu Thr Gly Lys AlaPhe Arg 50 55 60 Ile Gln Gly Ala Ala Ser Ser Leu Ser Asp Asp Ala Thr LeuPro Gly 65 70 75 80 Gly Ile Leu Arg Phe Ser Ser Leu Leu Val Ser Asn GlyTyr Ile Val 85 90 95 Val Asp Lys Tyr Phe Pro Arg Asn Thr Asn Ile Thr IleLys Asp Ala 100 105 110 Ser Gly Thr Val Ala Ala Gly Met Pro Phe Ile AspAla Asn Thr Ala 115 120 125 Ile Tyr Ser Asp Gln Leu Ser Ile Val Val ThrAsp Ser Thr Leu Ser 130 135 140 Trp Ala Ala Ala Arg Ser Gly Gln Ser MetVal Arg Ala Pro Phe Thr 145 150 155 160 Ile Gln Leu Ser Ser Ser Leu PheVal Leu Gly Ser Thr Val Thr Gln 165 170 175 Ala Ser Ser Val Val Glu ValVal Gly Pro Ser Ser Ile Ser Gln Lys 180 185 190 Ser Ala Leu Ala Val AspTyr Ala Lys Cys Thr Gly Cys Ala Gln Gly 195 200 205 Leu Val His Phe ThrAsp Phe Val Arg Val Trp Asp Arg Ser Leu Leu 210 215 220 Arg Val Ser HisSer Ser Val Lys Gly Ala Thr Gly Lys Pro Leu Ile 225 230 235 240 Gly IleAla Gln Ser Ala Gly Ala Ser Leu Ala Val Glu Asn Ser Leu 245 250 255 PheVal Val Glu Asn Val Ser Ser Pro Thr Ser Asn Leu Ile Asp Ala 260 265 270Ala Val Arg Met Gly Thr Asp Ala Gln Ile Thr Leu Arg Ala Val Thr 275 280285 Val Lys Ser Ile Gly Ala Thr Met Ala Gly Ser Val Thr Ala Gln Leu 290295 300 Leu Thr Ala Asp Asp Ile Ala Gln Gln Ile Pro Ser Ile Ser Val Val305 310 315 320 Pro Asp Thr Arg Cys Ala Ala Ala Cys Val Pro Thr Ala ThrVal Asp 325 330 335 Ser Ser Cys Lys Cys Ala Cys Asn Ala Asp Met Pro AsnMet Asn Phe 340 345 350 Cys Thr Ala Met Lys Asp Pro Tyr Thr Asn Tyr AlaTyr Leu Gly Cys 355 360 365 Ser Ala Gly Cys Thr Thr Cys Phe Asn Glu ThrAla Cys Leu Glu Cys 370 375 380 Arg Pro Ser Tyr Glu Met Leu Pro Asp MetThr Cys Ser Leu Thr Gly 385 390 395 400 Leu Gln Cys Thr Asp Pro Asn CysLys Thr Cys Thr Thr Tyr Gly Gln 405 410 415 Cys Thr Asp Cys Asn Asp GlyTyr Gly Leu Thr Ser Ser Ser Val Cys 420 425 430 Val Arg Cys Ser Val AlaGly Cys Lys Ser Cys Pro Val Asp Ala Asn 435 440 445 Val Cys Lys Val CysLeu Gly Gly Ser Glu Pro Ile Asn Asn Met Cys 450 455 460 Pro Cys Thr AspPro Asn Cys Ala Ser Cys Pro Ser Asp Ala Gly Thr 465 470 475 480 Cys ThrGln Cys Ala Asn Gly Tyr Gly Leu Val Asp Gly Ala Cys Val 485 490 495 ArgCys Gln Glu Pro Asn Cys Phe Ser Cys Asp Ser Asp Ala Asn Lys 500 505 510Cys Thr Gln Cys Ala Pro Asn Tyr Tyr Leu Thr Pro Leu Leu Thr Cys 515 520525 Ser Pro Val Ala Cys Asn Ile Glu His Cys Met Gln Cys Asp Pro Gln 530535 540 Thr Pro Ser Arg Cys Gln Glu Cys Val Ser Pro Tyr Val Val Asp Ser545 550 555 560 Tyr Asp Gly Leu Cys Arg Leu Ser Asp Ala Cys Ser Val ProAsn Cys 565 570 575 Lys Lys Cys Glu Thr Gly Thr Ser Arg Leu Cys Ala GluCys Asp Thr 580 585 590 Gly Tyr Ser Leu Ser Ala Asp Ala Thr Ser Cys SerSer Pro Thr Thr 595 600 605 Gln Pro Cys Glu Val Glu His Cys Asn Thr CysVal Asn Gly Asp Ser 610 615 620 Thr Arg Cys Ala Tyr Cys Asn Thr Gly TyrTyr Val Ser Asp Gly Lys 625 630 635 640 Cys Lys Ala Met Gln Gly Cys TyrVal Ser Asn Cys Ala Gln Cys Met 645 650 655 Leu Leu Asp Ser Thr Lys CysSer Thr Cys Val Lys Gly Tyr Leu Leu 660 665 670 Thr Ser Ser Tyr Ser CysVal Ser Gln Lys Val Ile Asn Ser Ala Ala 675 680 685 Ala Pro Tyr Ser LeuTrp Val Ala Ala Ala Val Leu Leu Thr Ser Phe 690 695 700 Ala Met His LeuAla 705 <210> SEQ ID NO 122 <211> LENGTH: 302 <212> TYPE: PRT <213>ORGANISM: Leishmania major and chagasi <400> SEQUENCE: 122 Met His HisHis His His His Asp Ser Gln Glu Ile Gly Gly Ser Ser 5 10 15 Phe Thr PheVal Gly Trp Ser Ser Ala Ser Lys Glu Glu Ser Tyr Gln 20 25 30 Gly Cys ThrLeu Thr Gly Lys Ala Phe Arg Ile Gln Gly Ala Ala Ser 35 40 45 Ser Leu SerAsp Asp Ala Thr Leu Pro Gly Gly Ile Leu Arg Phe Ser 50 55 60 Ser Leu LeuVal Ser Asn Gly Tyr Ile Val Val Asp Lys Tyr Phe Pro 65 70 75 80 Arg AsnThr Asn Ile Thr Ile Lys Asp Ala Ser Gly Thr Val Ala Ala 85 90 95 Gly MetPro Phe Ile Asp Ala Asn Thr Ala Ile Tyr Ser Asp Gln Leu 100 105 110 SerIle Val Val Thr Asp Ser Thr Leu Ser Trp Ala Ala Ala Arg Ser 115 120 125Gly Gln Ser Met Val Arg Ala Pro Phe Thr Ile Gln Leu Ser Ser Ser 130 135140 Leu Phe Val Leu Gly Ser Thr Val Thr Gln Ala Ser Ser Val Val Glu 145150 155 160 Val Val Gly Pro Ser Ser Ile Ser Gln Lys Ser Ala Leu Ala ValAsp 165 170 175 Tyr Ala Lys Cys Thr Gly Cys Ala Gln Gly Leu Val His PheThr Asp 180 185 190 Phe Val Arg Val Trp Asp Arg Ser Leu Leu Arg Val SerHis Ser Ser 195 200 205 Val Lys Gly Ala Thr Gly Lys Pro Leu Ile Gly IleAla Gln Ser Ala 210 215 220 Gly Ala Ser Leu Ala Val Glu Asn Ser Leu PheVal Val Glu Asn Val 225 230 235 240 Ser Ser Pro Thr Ser Asn Leu Ile AspAla Ala Val Arg Met Gly Thr 245 250 255 Asp Ala Gln Ile Thr Leu Arg AlaVal Thr Val Lys Ser Ile Gly Ala 260 265 270 Thr Met Ala Gly Ser Val ThrAla Gln Leu Leu Thr Ala Asp Asp Ile 275 280 285 Ala Gln Gln Ile Pro SerIle Ser Val Val Pro Asp Thr Arg 290 295 300

1. An isolated polypeptide comprising an immunogenic portion of aLeishmania antigen or a variant thereof, wherein said antigen comprisesan amino acid sequence selected from the group consisting of SEQ ID NO:2, 4, 20, 22, 24, 26, 36-38, 41, 50-53, 82, 104, 106, 108, 110, 112 and118-122, and variants thereof.
 2. An isolated antigenic epitope of aLeishmania antigen comprising an amino acid sequence selected from thegroup consisting of: SEQ ID NO: 43, 56, 57 or
 58. 3. An isolatedpolypeptide comprising at least two contiguous antigenic epitopesaccording to claim
 2. 4. An isolated polynucleotide comprising a DNAsequence encoding a polypeptide according to any one of claims 1 and 3.5. The polynucleotide of claim 4, wherein the DNA sequence is selectedfrom the group consisting of: SEQ ID NO: 1, 3, 19, 21, 23, 25, 29-31,34, 45-48, 74, 102, 103, 105, 107, 109, 111 and 113-117.
 6. Arecombinant expression vector comprising a polynucleotide according toclaim
 5. 7. A host cell transformed with an expression vector accordingto claim
 6. 8. The host cell of claim 7 wherein the host cell isselected from the group consisting of E. coli, yeast and mammaliancells.
 9. A fusion protein comprising at least two polypeptidesaccording to any one of claims 1 and
 3. 10. A fusion protein comprisingat least two antigenic epitopes according to claim
 2. 11. Apharmaceutical composition comprising a polypeptide according to any oneof claims 1 and 3, and a physiologically acceptable carrier.
 12. Apharmaceutical composition comprising a fusion protein according to anyone of claims 9 and 10, and a physiologically acceptable carrier
 13. Animmunogenic composition comprising a polypeptide according to any one ofclaims 1 and 3 and an immunostimulant.
 14. An immunogenic compositionaccording to claim 13 further comprising a delivery vehicle.
 15. Theimmunogenic composition of claim 14, wherein the delivery vehicle is abiodegradable microsphere.
 16. An immunogenic composition comprising apolynucleotide according to claim 4 and an immunostimulant.
 17. Animmunogenic composition comprising a fusion protein according to any oneof claims 9 and 10 and an immunostimulant
 18. A method for inducingprotective immunity against leishmaniasis in a patient comprisingadministering a pharmaceutical composition according to any one ofclaims 11 and
 12. 19. A method for inducing protective immunity againstleishmaniasis in a patient comprising administering an immunogeniccomposition according to any one of claims 13, 16 and
 17. 20. A methodfor detecting Leishmania infection in a patient, comprising: (a)contacting dermal cells of the patient with a pharmaceutical compositionaccording to any one of claims 11 and 12; and (b) detecting an immuneresponse on the patient's skin.
 21. The method of claim 20, wherein theimmune response is induration.
 22. A diagnostic kit comprising: (a) apharmaceutical composition according to any one of claim 11 and 12; and(b) apparatus sufficient to contact dermal cells of a patient with thepharmaceutical composition.
 23. The composition of claim 17, wherein theimmunostimulant is selected from the group consisting of: aminoalkylglucosaminide 4-phosphates; monophosphoryl lipid A; and 3-de-O-acylatedmonophosphoryl lipid A.